Apparatus for manipulating droplets

ABSTRACT

Apparatuses for mixing droplets, such as a binary mixing apparatus, are provided. The binary mixing apparatus includes an array of electrodes and a conducting element positioned in relation to at least one of the electrodes to enable a droplet placed in electrical communication with the at least one electrode to electrically communicate with the conducting element. The binary mixing apparatus additionally includes an electrode selector for sequentially biasing one or more selected electrodes of the array to move a droplet disposed on the array into contact with another droplet. The apparatus further includes a first droplet supply area communicating with the array and a second droplet supply area communicating with the array.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/253,342 filed Sep. 24, 2002; now U.S. Pat. No. 7,329,545 thedisclosure of which is incorporated by reference in its entirety.

GOVERNMENT INTEREST

This invention was made with Government support under Grant No.F30602-98-2-0140 awarded by the Defense Advanced Research ProjectsAgency. The Government has certain rights in the invention.

TECHNICAL FIELD

The present invention is generally related to the field of droplet-basedliquid handling and processing, such as droplet-based samplepreparation, mixing, and dilution on a microfluidic scale. Morespecifically, the present invention relates to apparatuses for mixingdroplets.

BACKGROUND ART

Microfluidic systems are presently being explored for their potential tocarry out certain processing techniques on capillary-sized continuousflows of liquid. In particular, there is currently great interest indeveloping microfluidic devices commonly referred to as“chemistry-on-a-chip” sensors and analyzers, which are also known aslabs-on-a-chip (LoC) and micro total analysis systems (μ-TAS). Theultimate goal of research in this field is to reduce most common(bio)chemical laboratory procedures and equipment to miniaturized,automated chip-based formats, thereby enabling rapid, portable,inexpensive, and reliable (bio)chemical instrumentation. Applicationsinclude medical diagnostics, environmental monitoring, and basicscientific research.

On-line monitoring of continuous flows is most often accomplished byconnecting the output of the continuous-flow to the input of a largeanalysis instrument such as a HPLC (high pressure liquidchromatography), CE (capillary electrophoresis) or MS (massspectrometry) system, with appropriate flow control and valving forsample collection and injection. Microfluidic systems for continuousmonitoring typically employ miniaturized analyte-specific biosensorswhere the continuous-flow stream passes over or through a series of thebiosensors. Because the sensors lie in a common channel, crosstalk orcontamination between sensors is often a concern. In analyses where areagent must be mixed with the flow, only one analyte can be measured ata time unless the flow is divided into parallel streams with separatemeans for adding the reagent, controlling and mixing the flow andcarrying out detection in each stream. Additionally, mixing inmicrofluidic flows is usually quite challenging. Sufficient time anddistance must be provided for mixing, which places constraints on chipdesign and system flow rates.

In general, mixing is a fundamental process in chemical analysis andbiological applications. Mixing in microfluidic devices is a criticalstep in realizing a μTAS (micro total analysis system) or “lab on achip” system. In accordance with the present invention describedhereinbelow, it is posited that mixing in these systems could be usedfor pre-processing sample dilution or for reactions between sample andreagents in particular ratios. It is further posited that the ability tomix liquids rapidly while utilizing minimum chip area would greatlyimprove the throughput of such systems. The improved mixing would relyon two principles: the ability to either create turbulent, nonreversibleflow at such small scales or create multilaminates to enhance mixing viadiffusion.

Mixers can be broadly categorized into continuous-flow and droplet-basedarchitectures. A common limitation among all continuous-flow systems isthat fluid transport is physically confined to permanently etchedstructures, and additional mechanisms are required to enhance mixing.The transport mechanisms used are usually pressure-driven by externalpumps or electrokinetically-driven by high-voltage supplies. This inturn requires the use of valves and complex channeling, consumingvaluable real estate on a chip. These restrictions prevent thecontinuous-flow micro-mixer from becoming a truly self-contained,reconfigurable lab-on-a-chip. Whereas conventional continuous-flowsystems rely on a continuous liquid flow in a confined channel,droplet-based systems utilize discrete volumes of liquid. Both thecontinuous-flow and droplet-based architectures can be furtherclassified into passive and active mixers. In passive mixers, mixing ismediated through diffusion passively without any external energyinputted for the process. Active mixing, on the other hand, takesadvantage of external energy, through actuation of some sort, to createeither dispersed multilaminates or turbulence. In the microscopic world,effective mixing is a technical problem because it is difficult togenerate turbulent flow by mechanical actuation. The inertial forcesthat produce turbulence and the resulting large interfacial surfaceareas necessary to promote mixing are absent. Thus, mixing that dependson diffusion through limited interfacial areas is a limitation.

Recently, active mixing by acoustic wave (see Vivek et al., “Novelacoustic micromixer”, MEMS 2000 p. 668-73); ultrasound (see Yang et al.,“Ultrasonic micromixer for microfluidic systems”, MEMS 2000, p. 80); anda piezoelectrically driven, valveless micropump (see Yang et al.,“Micromixer incorporated with piezoelectrically driven valvelessmicropump”, Micro Total Analysis System '98, p. 177-180) have beenproposed, and their effectiveness has been demonstrated. Mixing byelectroosmotic flow has also been described in U.S. Pat. No. 6,086,243to Paul et al. Another mixing technique has been recently presented byemploying chaotic advection for mixing. See Lee et al., “Chaotic mixingin electrically and pressure driven microflows”, The 14^(th) IEEEworkshop on MEMS 2001, p. 483-485; Liu et al., “Passive Mixing in aThree-Dimensional Serpentine Microchannel”, J. of MEMS, Vol 9 (No. 2),p. 190-197 (June 2000); and Evans et al., “Planar laminar mixer”, Proc.of IEEE, The tenth annual workshop on Micro Electro Mechanical Systems(MEMS 97), p. 96-101 (1997). Lee et al. focus on employingdielectrophoretic forces or pressure to generate chaotic advection,while Liu et al. rely on the geometry of a microchannel to induce thesimilar advection. Evans et al. constructed a planar mixing chamber onthe side of which an asymmetrical source and sink generate a flow field,whereby small differences in a fluid particle's initial location leadsto large differences in its final location. This causes chaoticrearrangement of fluid particles, and thus the mixing two liquids. Mostrecently, a technique has been proposed that uses electrohydrodynamicconvection for active mixing. See Jin et al., “An active micro mixerusing electrohydrodynamic (EHD) convection for microfluidic-basedbiochemical analysis”, Technical Digest, Solid-State Sensor and ActuatorWorkshop, p. 52-55).

Molecular diffusion plays an important role in small Reynolds numberliquid flow. In general, diffusion speed increases with the increase ofthe contact surface between two liquids. The time required for moleculardiffusion increases in proposition to the square of the diffusiondistance. A fast diffusion mixer consisting of a simple narrowing of amixing channel has been demonstrated by Veenstra et al.,“Characterization method for a new diffusion mixer applicable in microflow injection analysis systems”, J. Micromech. Microeng., Vol. 9, pg.199-202 (1999). The primary approach for diffusion-based micromixing hasbeen to increase the interfacial area and to decrease the diffusionlength by interleaving two liquids. Interleaving is done by manipulatingthe structure's geometry. One approach is to inject one liquid intoanother through a micro nozzle array. See Miyake et al., “Micro mixerwith fast diffusion”, Proceedings of Micro Electro Mechanical Systems,p. 248-253 (1993). An alternative method is to stack two flow streams inone channel as thin layers by multiple stage splitting and recombining.See Branebjerg et al., “Fast mixing by lamination”, Proc. IEEE MicroElectro Mechanical Systems, p. 441 (1996); Krog et al., “Experiments andsimulations on a micro-mixer fabricated using a planar silicon/glasstechnology”, MEMS, p. 177-182 (1998); Schwesinger et al., “A modularmicrofluidic system with an integrated micromixer”, J. Micromech.Microeng., Vol 6, pg. 99-102 (1996); and Schwesinger et al., “A staticmicromixer built up in silicon”, Proceedings of the SPIE, TheInternational Society for Optical Engineering, Micromachined Devices andComponents, Vol. 2642, p. 150-155. The characterizations of this type ofmixer are provided by Koch et al., “Two simple micromixers based onsilicon”, J. Micromech. Microeng., Vol 8, p. 123-126 (1998); Koch etal., “Micromachined chemical reaction system”, Sensors and Actuators,Physical (74), p. 207-210; and Koch et al., “Improved characterizationtechnique for micromixer, J. Micromech. Microeng, Vol 9, p. 156-158(1999). A variation of the lamination technique is achieved similarly byfractionation, re-arrangement, and subsequent reunification of liquidsin sinusoidally shaped fluid channels (see Kamper et al., “Microfluidiccomponents for biological and chemical microreactors”, MEMS 1997, p.338); in alternative channels of two counter current liquids; or in a 3Dpipe with a series of stationary rigid elements forming intersectingchannels inside (see Bertsch et al., “3D micromixers-downscaling largescale industrial static mixers”, MEMS 2001 14^(th) InternationalConference on Micro Electro Mechanical Systems, p. 507-510). Onedisadvantage of purely diffusion-based static mixing is the requirementof a complex 3D structure in order to provide out-of-plane fluid flow.Another disadvantage is the low Reynolds number characterizing the flow,which results in a long mixing time.

A problem for active mixers is that energy absorption during the mixingprocess makes them inapplicable to temperature-sensitive fluids.Moreover, some active mixers rely on the charged or polarizable fluidparticles to generate convection and local turbulence. Thus, liquidswith low conductivity could not be properly mixed. When the perturbationforce comes from a mechanical micropump, however, the presence of thevalveless micropump makes the control of flow ratios of solutions formixing quite complex.

In continuous flow systems, the control of the mixing ratio is always atechnical problem. By varying the sample and reagent flow rates, themixing ratio can be obtained with proper control of the pressure at thereagent and sample ports. However, the dependence of pressure on theproperties of the fluid and the geometry of the mixing chamber/channelsmakes the control very complicated. When inlets are controlled by amicropump, the nonlinear relationship between the operating frequencyand flow rate make it a nontrivial task to change the flow rate freely.The discontinuous mixing of two liquids by integration of a mixer and anelectrically actuated flapper valve has been demonstrated by Voldman etal., “An Integrated Liquid Mixer/Valve”, Journal ofMicroelectromechanical Systems”, Vol. 9, No. 3 (September 2000). Thedesign required a sophisticated pressure-flow calibration to get a rangeof mixing ratios.

Droplet-based mixers have been explored by Hosokawa et al., “Dropletbased nano/picoliter mixer using hydrophobic microcapillary vent”, MEMS'99, p. 388; Hosokawa et al., “Handling of Picoliter Liquid Samples in aPoly(dimethylsiloxane)-Based Microfluidic Device”, Anal. Chem 1999, Vol.71, p. 4781-4785; Washizu et al., Electrostatic actuation of liquiddroplets for micro-reactor applications, IEEE Transactions on IndustryApplications, Vol. 34 (No. 4), p. 732-737 (1998); Burns et al., “AnIntegrated Nanoliter DNA Analysis Device”, Science, Vol. 282 (No. 5388),p. 484 (Oct. 16, 1998); Pollack et al., “Electrowetting-based actuationof liquid droplets for microfluidic applications”, Appl. Phys. Lett.,Vol. 77, p. 1725 (September 2000); Pamula et al., “Microfluidicelectrowetting-based droplet mixing”, MEMS Conference, 2001, 8-10.;Fowler et al., “Enhancement of Mixing by Droplet-based Microfluidics”,IEEE MEMS Proceedings, 2002, 97-100.; Pollack, “Electrowetting-basedmicroactuation of droplets for digital microfluidics”, Ph.D. Thesis,Department of Electrical and Computer Engineering, Duke University; andWu, “Design and Fabrication of an Input Buffer for a Unit FlowMicrofluidic System”, Master thesis, Department of Electrical andComputer Engineering, Duke University.

It is believed that droplet-based mixers can be designed and constructedto provide a number of advantages over continuous-flow-basedmicrofluidic devices. Discrete flow can eliminate the limitation on flowrate imposed by continuous microfluidic devices. The design ofdroplet-based mixing devices can be based on a planar structure that canbe fabricated at low cost. Actuation mechanisms based on pneumaticdrive, electrostatic force, or electrowetting do not require heaters,and thus have a minimum effect on (bio) chemistry. By providing a properdroplet generation technique, droplet-based mixers can provide bettercontrol of liquid volume. Finally, droplet-based mixers can enabledroplet operations such as shuttling or shaking to generate internalrecirculation within the droplet, thereby increasing mixing efficiencyin the diffusion-dominated scale.

In view of the foregoing, it would be advantageous to provide noveldroplet-manipulative techniques to address the problems associated withprevious analytical and mixing techniques that required continuousflows. In particular, the present invention as described and claimedhereinbelow developed in part from the realization that an alternativeand better solution to the continuous flow architecture would be todesign a system where the channels and mixing chambers are notpermanently etched, but rather are virtual and can be configured andreconfigured on the fly. The present invention enables such a system byproviding means for discretizing fluids into droplets and means forindependently controlling individual droplets, allowing each droplet toact as a virtual mixing or reaction chamber.

DISCLOSURE OF THE INVENTION

The present invention provides droplet-based liquid handling andmanipulation methods by implementing electrowetting-based techniques.The droplets can be sub-microliter-sized, and can be moved freely bycontrolling voltages to electrodes. Generally, the actuation mechanismof the droplet is based upon surface tension gradients induced in thedroplet by the voltage-induced electrowetting effect. The mechanisms ofthe invention allow the droplets to be transported while also acting asvirtual chambers for mixing to be performed anywhere on a chip. The chipcan include an array of electrodes that are reconfigurable in real-timeto perform desired tasks. The invention enables several different typesof handling and manipulation tasks to be performed on independentlycontrollable droplet samples, reagents, diluents, and the like. Suchtasks conventionally have been performed on continuous liquid flows.These tasks include, for example, actuation or movement, monitoring,detection, irradiation, incubation, reaction, dilution, mixing,dialysis, analysis, and the like. Moreover, the methods of the inventioncan be used to form droplets from a continuous-flow liquid source, suchas a from a continuous input provided at a microfluidic chip.Accordingly, the invention provides a method for continuous sampling bydiscretizing or fragmenting a continuous flow into a desired number ofuniformly sized, independently controllable droplet units.

The partitioning of liquids into discrete, independently controlledpackets or droplets for microscopic manipulation provides severalimportant advantages over continuous-flow systems. For instance, thereduction of fluid manipulation, or fluidics, to a set of basic,repeatable operations (for example, moving one unit of liquid one unitstep) allows a hierarchical and cell-based design approach that isanalogous to digital electronics.

In addition to the advantages identified hereinabove, the presentinvention utilizes electrowetting as the mechanism for droplet actuationor manipulation for the following additional advantages:

1. Improved control of a droplet's position.

2. High parallelism capability with a dense electrode array layout.

3. Reconfigurability.

4. Mixing-ratio control using programming operations, yielding bettercontrollability and higher accuracy in mixing ratios.

5. High throughput capability, providing enhanced parallelism.

6. Enabling of integration with optical detection that can providefurther enhancement on asynchronous controllability and accuracy.

In particular, the present invention provides a sampling method thatenables droplet-based sample preparation and analysis. The presentinvention fragments or discretizes the continuous liquid flow into aseries of droplets of uniform size on or in a microfluidic chip or othersuitable structure by inducing and controlling electrowetting phenomena.The liquid is subsequently conveyed through or across the structure as atrain of droplets which are eventually recombined for continuous-flow atan output, deposited in a collection reservoir, or diverted from theflow channel for analysis. Alternatively, the continuous-flow stream maycompletely traverse the structure, with droplets removed or sampled fromspecific locations along the continuous flow for analysis. In bothcases, the sampled droplets can then be transported to particular areasof the structure for analysis. Thus, the analysis is carried outon-line, but not in-line with respect to the main flow, allowing theanalysis to be de-coupled from the main flow.

Once removed from the main flow, a facility exists for independentlycontrolling the motion of each droplet. For purposes of chemicalanalysis, the sample droplets can be combined and mixed with dropletscontaining specific chemical reagents formed from reagent reservoirs onor adjacent to the chip or other structure. Multiple-step reactions ordilutions might be necessary in some cases with portions of the chipassigned to certain functions such as mixing, reacting or incubation ofdroplets. Once the sample is prepared, it can be transported byelectrowetting to another portion of the chip dedicated to detection ormeasurement of the analyte. Some detection sites can, for example,contain bound enzymes or other biomolecular recognition agents, and bespecific for particular analytes while others can consist of a generalmeans of detection such as an optical system for fluorescence orabsorbance based assays. The flow of droplets from the continuous flowsource to the analysis portion of the chip (the analysis flow) iscontrolled independently of the continuous flow (the input flow),allowing a great deal of flexibility in carrying out the analyses. Otherfeatures and advantages of the methods of the present invention aredescribed in more detail hereinbelow.

Methods of the present invention use means for forming microdropletsfrom the continuous flow and for independently transporting, merging,mixing, and other processing of the droplets. The preferred embodimentuses electrical control of surface tension (i.e., electrowetting) toaccomplish these manipulations. In one embodiment, the liquid iscontained within a space between two parallel plates. One plate containsetched drive electrodes on its surface while the other plate containseither etched electrodes or a single, continuous plane electrode that isgrounded or set to a reference potential. Hydrophobic insulation coversthe electrodes and an electric field is generated between electrodes onopposing plates. This electric field creates a surface-tension gradientthat causes a droplet overlapping the energized electrode to movetowards that electrode. Through proper arrangement and control of theelectrodes, a droplet can be transported by successively transferring itbetween adjacent electrodes. The patterned electrodes can be arranged ina two dimensional array so as to allow transport of a droplet to anylocation covered by that array. The space surrounding the droplets maybe filled with a gas such as air or an immiscible fluid such as oil.

In another embodiment, the structure used for ground or referencepotential is co-planar with the drive electrodes and the second plate,if used, merely defines the containment space. The co-planar groundingelements can be a conductive grid superimposed on the electrode array.Alternatively, the grounding elements can be electrodes of the arraydynamically selected to serve as ground or reference electrodes whileother electrodes of the array are selected to serve as drive electrodes.

Droplets can be combined together by transporting them simultaneouslyonto the same electrode. Droplets are subsequently mixed eitherpassively or actively. Droplets are mixed passively by diffusion.Droplets are mixed actively by moving or “shaking” the combined dropletby taking advantage of the electrowetting phenomenon. In a preferredembodiment, droplets are mixed by rotating them around a two-by-twoarray of electrodes. The actuation of the droplet creates turbulentnon-reversible flow, or creates dispersed multilaminates to enhancemixing via diffusion. Droplets can be split off from a larger droplet orcontinuous body of liquid in the following manner: at least twoelectrodes adjacent to the edge of the liquid body are energized alongwith an electrode directly beneath the liquid, and the liquid moves soas to spread across the extent of the energized electrodes. Theintermediate electrode is then de-energized to create a hydrophobicregion between two effectively hydrophilic regions. The liquid meniscusbreaks above the hydrophobic regions, thus forming a new droplet. Thisprocess can be used to form the droplets from a continuously flowingstream.

According to one embodiment of the present invention, a method forsampling a continuous liquid flow comprises the following steps. Aliquid flow is supplied to a surface along a first flow path. The liquidflow is sampled by forming a sample droplet from a portion of the liquidflow. The sample droplet is moved along a second flow path to aprocessing area of the surface. The sample droplet is processed at theprocessing area.

In one aspect of this method, the liquid flow is sampled by sequentiallyenergizing and de-energizing a selected set of the electrodes of anarray associated with the surface. One of the selected electrodes isdisposed under the liquid flow, another electrode is adjacent to an edgeof the liquid flow, and a third electrode is adjacent to the secondelectrode. Additional electrodes of the array can be sequentiallyenergized and de-energized in order to move the sample droplet along anintended analysis flow path, such as for transporting the sample dropletto the processing area. In certain embodiments of the invention, theprocessing area is configured on a real time basis by sequentialenergizing and de-energizing of a selected set of the electrodes of thearray. For example, through appropriate sequential activation of certainelectrodes on the array, preferably by controlling the electrodes inthis area through communication with an electronic controller thatexecutes a preprogrammed set of instructions, a functional device suchas a mixer can be created.

In one embodiment, the mixer is defined at least in part by a firstelectrode, a second electrode adjacent to the first electrode, and athird electrode adjacent to the second electrode. The sample droplet isinitially positioned at the first electrode, and an additional dropletcontaining a diluent or reagent material is initially positioned at thethird electrode. The sample droplet is combined with the additionaldroplet by moving the sample droplet from the first electrode to theintermediate second electrode and moving the additional droplet from thethird electrode to the second electrode, whereby the combined droplet isformed on the second electrode. In accordance with the invention, thesemanipulations of the droplets are effected by taking advantage of theelectrowetting technique disclosed herein. Thus, all three electrodesare energized such that the sample droplet and the additional dropletare drawn onto the intermediate second electrode. The first and thirdelectrodes are then de-energized, with the result that the combineddroplet is centered on the second electrode.

In many cases, it is desirable to maintain uniformity in droplet sizeduring the course of carrying out operations on the array. Accordingly,the combined droplet can be split by energizing all three electrodes tocause the combined droplet to spread to the first and third electrodes,and thereafter de-energizing the intermediate second electrode to breakthe meniscus of the combined droplet. As a result, a first mixed dropletis formed on the first electrode and a second mixed droplet is formed onthe third electrode, with each mixed droplet containing components ofthe other droplet. This process is useful for diluting the initiallyprovided sample droplet to a desired concentration.

According to another method for sampling a continuous liquid flow, aliquid flow is supplied to a surface along an input flow path. Theliquid flow is sampled by forming a first sample droplet and a secondsample droplet from a portion of the liquid flow. The first sampledroplet is moved along a first analysis flow path to a first processingarea of the surface. The second sample droplet is moved along a secondanalysis flow path to a second processing area of the surface. The firstsample droplet can then be processed at the first processing area andthe second sample droplet processed at the second processing area. Therespective processing rates of the first and second sample droplets canbe controlled, as well as their movement rates. Moreover, the types ofprocesses conducted on the first and second sample droplets can be thesame or different. For example, the first sample droplet can be combinedor reacted with a reagent droplet of a first type, while the secondsample droplet is combined or reacted with a reagent of a second type.In another example, the first sample droplet is subjected to a mixingprocess, while the second sample droplet is subjected to a detection ormeasuring process. In this latter case, the second sample droplet couldbe used as a reference, blank or control sample for calibration purposesso as to increase the accuracy of the detection or measurement of thefirst sample droplet.

According to yet another embodiment of the invention, a method isprovided for performing microfluidic sampling. The method comprises thefollowing steps. A substrate is provided that comprises an arrangementof at least first, second and third control electrodes. The secondcontrol electrode is interposed between and adjacent to the first andthird control electrodes. A fluid input flow stream is caused to flow tothe first control electrode. The first, second and third controlelectrodes are energized to cause a portion of the fluid input flowstream to spread across the second and third control electrodes. Thesecond electrode is de-energized to form a droplet on the third controlelectrode, which droplet is separate from the fluid input flow stream.This method allows any further manipulation of the droplet to becontrolled independently of the fluid input flow stream. According toone aspect of this method, the fluid input flow stream is sequentiallyfragmented into a train of one or more droplets. According to analternative aspect of this method, the fluid input flow stream ispermitted to flow across the liquid handling region or a portionthereof, and the droplet or droplets formed from this fluid input flowstream are directed away from the direction of the fluid input flowstream.

According to still another embodiment of the invention, a method forsampling a continuous liquid flow comprises supplying a liquid flow to asurface along a first flow path and sampling the liquid flow byperforming an electrowetting technique. Use of the electrowettingtechnique forms a sample droplet on the surface that is distinct fromthe liquid flow and controllable independently of the liquid flow. Afterthe sample droplet is formed, it can be moved along a second flow pathby electrowetting.

According to additional embodiments of the present invention, methodsare provided for mixing the contents of a droplet. In a preferredmethod, a two-by-two array is provided that comprises first, second,third, and fourth electrodes. A multi-component droplet comprising afirst composition and a second composition to be mixed is placed inelectrical communication with the first, second, third, and fourthelectrodes and also with a grounded element. The first and secondcompositions of the multi-component droplet are mixed by sequentiallyenergizing and de-energizing the first, second, third, and fourthelectrodes to cause the multi-component droplet to rotate. The dropletcan be placed in electrical communication with all four of theelectrodes by being sized such that the droplet overlaps all fourelectrodes. Thus, in effect, a portion of the droplet remainssubstantially stationary at or near an intersecting region between thefour electrodes while the droplet rotates. In this manner, the actuationof the droplet can be characterized as spinning as a result of thesequencing of the four electrodes. In many applications, themulti-component droplet is formed by first combining one dropletcomprising the first composition with another droplet comprising thesecond composition.

The present invention also provides a method and apparatus for digitallymixing the contents of two or more liquids together in a series ofbinary operations to form a mixture of a predetermined ratio. Thisaspect of the present invention entails rapid droplet-to-droplet mixingthat exploits electrowetting technology in handling discrete liquidamounts on a flexible and reconfigurable electrode array used fordroplet transport. The method takes full advantage of accelerated ratesobserved in droplet-to-droplet mixing while allowing preciselycontrolled mixing ratios that could be varied dynamically formulti-point calibrations. A preferred strategy is to use multiplehierarchies of binary mixing phases, with the first hierarchy providingthe approximate mixing ratio and the following ones employed as thecalibration mechanism. The concept is analogous to an interpolatingDigital-to-Analog Converter (DAC) whose architecture is divided into twoparts, with the main DAC handling the MSB (most significant bit) in abinary manner and the sub-DAC dealing with calibration and correction.

In one embodiment of the invention, a binary mixing apparatus comprisesan array, an electronic controller, a sample droplet supply areacommunicating with the array, and a diluent droplet supply areacommunicating with the array. The array comprises a plurality of controlelectrodes. The electronic controller communicates with the controlelectrodes and is adapted for causing selected control electrodes to beenergized. Sequencing of the control electrodes can be accomplishedaccording to a programmable computer program instructions executed bythe electronic controller. Preferably, the array comprises a first plateon which the plurality of control electrodes are disposed, and a secondplate spaced in parallel with the first plate, at least one ground orreference electrode supported by the second plate and aligned with thecontrol electrodes. The first and second plates define a spacetherebetween. Preferably, a fluid is contained in the space to surroundany droplets processed by the binary mixing apparatus. This fluid canconstitute an inert gas such as air or an immiscible liquid such assilicone oil. Preferably, a first hydrophobic layer is disposed on thefirst plate to isolate the control electrodes, and a second hydrophobiclayer is disposed on the second plate to isolate the ground electrode.

In another embodiment of the invention, the binary mixing apparatuscomprises first and second mixing units comprising respective first andsecond arrays of control electrodes, and an electronic controllercommunicating with the first and second control electrodes. Each mixingunit is adapted for individually mixing sample droplets with diluent orreagent droplets to produce respective combined droplets having desiredrespective mixing ratios. These combined droplets can serve asintermediate diluted droplets. One of these intermediate droplets can betransported to the other mixing unit and combined with the otherintermediate droplet in order to produce a droplet having a finaldesired mixing ratio.

According to a method of the invention for producing a droplet having adesired mixing ratio, an array comprising electrodes is provided. Asample droplet having an initial concentration and a diluent droplet areprovided on the array. A selected set of the electrodes are sequentiallyenergized and de-energized to mix the sample droplet with the diluentdroplet to form a combined droplet having a reduced concentrationcorresponding to an approximate mixing ratio. The mixing step is thenrepeated for a determined number of times, using one or more additionaldiluent droplets to form one or more new combined droplets, until thereduced concentration of the last combined droplet approaches thedesired mixing ratio within a desired degree of accuracy. Preferably, asuitable combination of one or more row-to-row mixing steps and one ormore column-to-column mixing steps is utilized.

According to another method for producing a droplet having a desiredmixing ratio, an electrode array comprising a plurality of controlelectrodes is provided. A first droplet having a first composition isprovided on a first control electrode of the electrode array. A seconddroplet having a second composition is provided on a second controlelectrode of the electrode array. A third control electrode of theelectrode array, disposed between and adjacent to the first and secondcontrol electrodes, is energized to cause the first droplet and thesecond droplet to mix with each other at the third control electrode. Amixed droplet is formed at the third control electrode that comprises amixture of the first and second compositions.

According to yet another method of the present invention, a droplet isproduced that has a desired final mixing ratio. In a first mixing unit,a first sample droplet is mixed with a first diluent droplet to form afirst combined droplet having a desired first intermediate mixing ratio.In a second mixing unit, a second sample droplet is mixed with a seconddiluent droplet to form a second combined droplet having a desiredsecond intermediate mixing ratio. The second combined droplet istransported to the first mixing unit. In the first mixing unit, thefirst combined droplet is combined with the second combined droplet toform a third combined droplet having the desired final mixing ratio.

It is therefore an object of the present invention to sample acontinuous flow liquid input source from which uniformly sized,independently controllable droplets are formed on a continuous andautomated basis.

It is another object of the present invention to utilize electrowettingtechnology to implement and control droplet-based manipulations such astransportation, mixing, detection, analysis, and the like.

It is yet another object of the present invention to provide anarchitecture suitable for efficiently performing binary mixing ofdroplets to obtain desired mixing ratios with a high degree of accuracy.

Some of the objects of the invention having been stated hereinabove,other objects will become evident as the description proceeds when takenin connection with the accompanying drawings as best describedhereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of an electrowetting microactuatormechanism having a two-sided electrode configuration in accordance withthe present invention;

FIG. 2 is a top plan view of an array of electrode cells havinginterdigitated perimeters accordance with one embodiment of the presentinvention;

FIG. 3 is a plot of switching rate as a function of voltagedemonstrating the performance of an electrowetting microactuatormechanism structured in accordance with the present invention;

FIGS. 4A-4D are sequential schematic views of a droplet being moved bythe electrowetting technique of the present invention;

FIGS. 5A-5C are sequential schematic views illustrating two dropletscombining into a merged droplet using the electrowetting technique ofthe present invention;

FIGS. 6A-6C are sequential schematic views showing a droplet being splitinto two droplets by the electrowetting technique of the presentinvention;

FIGS. 7A and 7B are sequential schematic views showing a liquid beingdispensed on an electrode array and a droplet being formed from theliquid;

FIG. 8A is a cross-sectional view illustrating an electrowettingmicroactuator mechanism of the invention implementing a one-dimensionallinear droplet merging process;

FIG. 8B is a top plan view of the configuration in FIG. 8A with theupper plane removed;

FIGS. 9A, 9B, and 9C are respective top plan views of two-, three-, andfour-electrode configurations on which one-dimensional linear mixing ofdroplets can be performed in accordance with the present invention;

FIGS. 10A, 10B, and 10C are schematic diagrams illustrating the examplesof a mixing-in-transport process enabled by the present invention;

FIG. 11 is a schematic view illustrating a two-dimensional linear mixingprocess enabled by the present invention;

FIG. 12A is a top plan view of an array of electrode cells on which atwo-dimensional loop mixing process is performed in accordance with thepresent invention;

FIG. 12B is a top plan view of a 2×2 array of electrode cells on which atwo-dimensional loop mixing process is performed in which a portion ofthe droplet remains pinned during rotation;

FIG. 13 is a plot of data characterizing the performance of activedroplet mixing using the two-, three- and four-electrode configurationsrespectively illustrated in FIGS. 9A, 9B, and 9C;

FIG. 14 is a plot of data characterizing the performance of the 2×2electrode configuration illustrated in FIG. 12B;

FIG. 15A is a schematic view illustrating the formation of droplets froma continuous flow source and movement of the droplets across anelectrode-containing surface to process areas of the surface;

FIG. 15B is a schematic view illustrating the formation of droplets froma continuous flow that traverses an entire electrode-containing surfaceor section thereof;

FIG. 16 is a top plan view of a droplet-to-droplet mixing unit that canbe defined on an electrode array on a real-time basis;

FIG. 17 is a schematic view of a binary mixing apparatus provided inaccordance with the present invention;

FIG. 18A is a schematic view of the architecture of a binary mixing unitcapable of one-phase mixing according to the present invention;

FIG. 18B is a schematic sectional view of the binary mixing unitillustrated in FIG. 18A, showing details of the matrix section thereofwhere binary mixing operations occur;

FIGS. 19A-19F are sequential schematic views of an electrode array orsection thereof provided by a binary mixing unit of the presentinvention, showing an exemplary process for performing binary mixingoperations to obtain droplets having a predetermined, desired mixingratio;

FIG. 20 is a schematic view illustrating the architecture for a binarymixing unit capable of two-phase mixing in accordance with the presentinvention;

FIG. 21 is a plot of mixing points of a one- and two-phase mixing planenabled by the binary mixing architecture of the present invention; and

FIG. 22 is a plot of mixing points of a one-, two- and three-phasemixing plan enabled by the binary mixing architecture of the presentinvention.

FIG. 23A is a cross-sectional view of an electrowetting microactuatormechanism having a single-sided electrode configuration in accordancewith another embodiment of the present invention;

FIG. 23B is a top plan view of a portion of the mechanism illustrated inFIG. 23A with its upper plane removed;

FIGS. 24A-24D are sequential schematic views of an electrowettingmicroactuator mechanism having an alternative single-sided electrodeconfiguration, illustrating electrowetting-based movement of a dropletpositioned on a misaligned electrode array of the mechanism; and

FIGS. 25A and 25B are schematic views of an alternative electrowettingmicroactuator mechanism having a single-sided electrode configurationarranged as an aligned array, respectively illustrating a dropletactuated in north-south and east-west directions.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of the present disclosure, the terms “layer” and “film” areused interchangeably to denote a structure or body that is typically butnot necessarily planar or substantially planar, and is typicallydeposited on, formed on, coats, treats, or is otherwise disposed onanother structure.

For purposes of the present disclosure, the term “communicate” (e.g., afirst component “communicates with” or “is in communication with” asecond component) is used herein to indicate a structural, functional,mechanical, electrical, optical, or fluidic relationship, or anycombination thereof, between two or more components or elements. Assuch, the fact that one component is said to communicate with a secondcomponent is not intended to exclude the possibility that additionalcomponents may be present between, and/or operatively associated orengaged with, the first and second components.

For purposes of the present disclosure, it will be understood that whena given component such as a layer, region or substrate is referred toherein as being disposed or formed “on”, “in”, or “at” anothercomponent, that given component can be directly on the other componentor, alternatively, intervening components (for example, one or morebuffer layers, interlayers, electrodes or contacts) can also be present.It will be further understood that the terms “disposed on” and “formedon” are used interchangeably to describe how a given component ispositioned or situated in relation to another component. Hence, theterms “disposed on” and “formed on” are not intended to introduce anylimitations relating to particular methods of material transport,deposition, or fabrication.

For purposes of the present disclosure, it will be understood that whena liquid in any form (e.g., a droplet or a continuous body, whethermoving or stationary) is described as being “on”, “at”, or “over” anelectrode, array, matrix or surface, such liquid could be either indirect contact with the electrode/array/matrix/surface, or could be incontact with one or more layers or films that are interposed between theliquid and the electrode/array/matrix/surface.

As used herein, the term “reagent” describes any material useful forreacting with, diluting, solvating, suspending, emulsifying,encapsulating, interacting with, or adding to a sample material.

The droplet-based methods and apparatus provided by the presentinvention will now be described in detail, with reference being made asnecessary to the accompanying FIGS. 1-25B.

Droplet-Based Actuation by Electrowetting

Referring now to FIG. 1, an electrowetting microactuator mechanism,generally designated 10, is illustrated as a preferred embodiment foreffecting electrowetting-based manipulations on a droplet D without theneed for pumps, valves, or fixed channels. Droplet D is electrolytic,polarizable, or otherwise capable of conducting current or beingelectrically charged. Droplet D is sandwiched between a lower plane,generally designated 12, and an upper plane, generally designated 14.The terms “upper” and “lower” are used in the present context only todistinguish these two planes 12 and 14, and not as a limitation on theorientation of planes 12 and 14 with respect to the horizontal. Lowerplane 12 comprises an array of independently addressable controlelectrodes. By way of example, a linear series of three control or driveelectrodes E (specifically E₁, E₂, and E₃) are illustrated in FIG. 1. Itwill be understood, however, that control electrodes E₁, E₂, and E₃could be arranged along a non-linear path such as a circle. Moreover, inthe construction of devices benefiting from the present invention (suchas a microfluidic chip), control electrodes E₁, E₂, and E₃ willtypically be part of a larger number of control electrodes thatcollectively form a two-dimensional electrode array or grid. FIG. 1includes dashed lines between adjacent control electrodes E₁, E₂, and E₃to conceptualize unit cells, generally designated C (specifically C₁, C₂and C₃). Preferably, each unit cell C₁, C₂, and C₃ contains a singlecontrol electrode, E₁, E₂, and E₃, respectively. Typically, the size ofeach unit cell C or control electrode E is between approximately 0.05 mmto approximately 2 mm.

Control electrodes E₁, E₂, and E₃ are embedded in or formed on asuitable lower substrate or plate 21. A thin lower layer 23 ofhydrophobic insulation is applied to lower plate 21 to cover and therebyelectrically isolate control electrodes E₁, E₂, and E₃. Lowerhydrophobic layer 23 can be a single, continuous layer or alternativelycan be patterned to cover only the areas on lower plate 21 where controlelectrodes E₁, E₂ and E₃ reside. Upper plane 14 comprises a singlecontinuous ground electrode G embedded in or formed on a suitable uppersubstrate or plate 25. Alternatively, a plurality of ground electrodes Gcould be provided in parallel with the arrangement of correspondingcontrol electrodes E₁, E₂ and E₃, in which case one ground electrode Gcould be associated with one corresponding control electrode E.Preferably, a thin upper layer 27 of hydrophobic insulation is alsoapplied to upper plate 25 to isolate ground electrode G. Onenon-limiting example of a hydrophobic material suitable for lower layer23 and upper layer 27 is TEFLON® AF 1600 material (available from E. I.duPont deNemours and Company, Wilmington, Del.). The geometry ofmicroactuator mechanism 10 and the volume of droplet D are controlledsuch that the footprint of droplet D overlaps at least two controlelectrodes (e.g., E₁ and E₃) adjacent to the central control electrode(e.g., E₂) while also making contact with upper layer 27. Preferably,this is accomplished by specifying a gap or spacing d, which is definedbetween lower plane 12 and upper plane 14 as being less than thediameter that droplet D would have in an unconstrained state. Typically,the cross-sectional dimension of spacing d is between approximately 0.01mm to approximately 1 mm. Preferably, a medium fills gap d and thussurrounds droplet D. The medium can be either an inert gas such as airor an immiscible fluid such as silicone oil to prevent evaporation ofdroplet D.

Ground electrode G and control electrodes E₁, E₂ and E₃ are placed inelectrical communication with at least one suitable voltage source V,which preferably is a DC voltage source but alternatively could be an ACvoltage source, through conventional conductive lead lines L₁, L₂ andL₃. Each control electrode E₁, E₂ and E₃ is energizable independently ofthe other control electrodes E₁, E₂ and E₃. This can be accomplished byproviding suitable switches S₁, S₂ and S₃ communicating with respectivecontrol electrodes E₁, E₂ and E₃, or other suitable means forindependently rendering each control electrode E₁, E₂ and E₃ eitheractive (ON state, high voltage, or binary 1) or inactive (OFF state, lowvoltage, or binary 0). In other embodiments, or in other areas of theelectrode array, two or more control electrodes E can be commonlyconnected so as to be activated together.

The structure of electrowetting microactuator mechanism 10 can representa portion of a microfluidic chip, on which conventional microfluidicand/or microelectronic components can also be integrated. As examples,the chip could also include resistive heating areas, microchannels,micropumps, pressure sensors, optical waveguides, and/or biosensing orchemosensing elements interfaced with MOS (metal oxide semiconductor)circuitry.

Referring now to FIG. 2, an electrode array or portion thereof isillustrated in which each structural interface between adjacent unitcells (e.g., C₁ and C₂) associated with control electrodes (not shown)is preferably characterized by an interdigitated region, generallydesignated 40, defined by interlocking projections 42 and 43 extendingoutwardly from the main planar structures of respective unit cells C₁and C₂. Such interdigitated regions 40 can be useful in rendering thetransition from one unit cell (e.g., C₁) to an adjacent unit cell (e.g.,C₂) more continuous, as opposed to providing straight-edged boundariesat the cell-cell interfaces. It will be noted, however, that theelectrodes or unit cells according to any embodiment of the inventioncan have any polygonal shape that is suitable for constructing aclosely-packed two-dimensional array, such as a square or octagon.

Referring back to FIG. 1, the basic electrowetting technique enabled bythe design of microactuator mechanism 10 will now be described.Initially, all control electrodes (i.e., control electrode E₂ on whichdroplet D is centrally located and adjacent control electrodes E₁ andE₃) are grounded or floated, and the contact angle everywhere on dropletD is equal to the equilibrium contact angle associated with that dropletD. When an electrical potential is applied to control electrode E₂situated underneath droplet D, a layer of charge builds up at theinterface between droplet D and energized control electrode E₂,resulting in a local reduction of the interfacial energy γ_(SL). Sincethe solid insulator provided by lower hypdrophobic insulating layer 23controls the capacitance between droplet D and control electrode E₂, theeffect does not depend on the specific space-charge effects of theelectrolytic liquid phase of droplet D, as is the case in previouslydeveloped uninsulated electrode implementations.

The voltage dependence of the interfacial energy reduction is describedby

$\begin{matrix}{{{\gamma_{SL}(V)} = {{\gamma_{SL}(O)} - {\frac{ɛ}{2d}V^{2}}}},} & (1)\end{matrix}$where ∈ is the permittivity of the insulator, d is the thickness of theinsulator, and V is the applied potential. The change in γ_(SL) actsthrough Young's equation to reduce the contact angle at the interfacebetween droplet D and energized control electrode E₂. If a portion ofdroplet D also overlaps a grounded electrode E₁ or E₃, the dropletmeniscus is deformed asymmetrically and a pressure gradient isestablished between the ends of droplet D, thereby resulting in bulkflow towards the energized electrode E₁ or E₃. For example, droplet Dcan be moved to the left (i.e., to unit cell C₁) by energizing controlelectrode E₁ while maintaining control electrodes E₂ and E₃ at theground state. As another example, droplet D can be moved to the right(i.e., to unit cell C₃) by energizing control electrode E₃ whilemaintaining control electrodes E₁ and E₂ at the ground state.

The following EXAMPLE describes a prototypical embodiment ofelectrowetting microactuator mechanism 10, with reference beinggenerally made to FIGS. 1 and 2.

EXAMPLE

A prototype device consisting of a single linear array of seveninterdigitated control electrodes E at a pitch of 1.5 mm was fabricatedand tested. Control electrodes E were formed by patterning a 2000-Åthick layer of chrome on a glass lower plate 21 using standardmicrofabrication techniques. The chips were then coated with a 7000 Ålayer of Parylene C followed by a layer 23 of approximately 2000 Å ofTEFLON® AF 1600. Ground electrode G consisted of an upper plate 25 ofglass coated with a conducting layer (R_(ζ)<20 Ω/square) of transparentindium-tin-oxide (ITO). A thin (˜500 Å) layer 27 of TEFLON® AF 1600 wasalso applied to ground electrode G. The thin TEFLON® coating on groundelectrode G served to hydrophobize the surface, but was not presumed tobe insulative. After coating with TEFLON®, both surfaces had a contactangle of 104° with water.

Water droplets (0.7-1.0 μl) of 100 mM KCl were dispensed onto the arrayusing a pipette, and upper plate 25 was positioned to provide a gap d of0.3 mm between the opposing electrodes E and G. A customized clamp withspring-loaded contact pins (not shown) was used to make connections tothe bond pads. A computer was used to control a custom-built electronicinterface which was capable of independently switching each outputbetween ground and the voltage output of a 120 V DC power supply.

A droplet D was initially placed on the center of the grounded controlelectrode (e.g., E₂) and the potential on the adjacent electrode (e.g.,control electrode E₁ or E₃) was increased until motion was observed.Typically, a voltage of 30-40 V was required to initiate movement ofdroplet D. Once this threshold was exceeded, droplet movement was bothrapid and repeatable. It is believed that contact angle hysteresis isthe mechanism responsible for this threshold effect. By sequentiallyenergizing four adjacent control electrodes E at 80 V of appliedpotential, droplet D was moved repeatedly back and forth across all fourcontrol electrodes E at a switching frequency of 15 Hz.

The transit time t_(tr) of the droplet D was defined as the timerequired for droplet D to reach the far edge of the adjacent electrodefollowing the application of the voltage potential. The transit timet_(tr) thus represented the minimum amount of time allowed betweensuccessive transfers, and (1/t_(tr)) was the maximum switching rate forcontinuous transfer of a droplet D. The maximum switching rate as afunction of voltage is plotted in FIG. 3, where t_(tr) was determined bycounting recorded video frames of a moving droplet D.

Sustained droplet transport over 1000's of cycles at switching rates ofup to 1000 Hz has been demonstrated for droplets of 6 nL volume. Thisrate corresponds to an average droplet velocity of 10.0 cm/s, which isnearly 300 times faster than a previously reported method for electricalmanipulation of droplets. See M. Washizu, IEEE Trans. Ind. Appl. 34, 732(1998). Comparable velocities cannot be obtained in thermocapillarysystems because (for water) the required temperature difference betweenthe ends of droplet D exceeds 100° C. See Sammarco et al., AIChE J., 45,350 (1999). These results demonstrate the feasibility of electrowettingas an actuation mechanism for droplet-based microfluidic systems. Thisdesign can be extended to arbitrarily large two-dimensional arrays toallow precise and independent control over large numbers of droplets Dand to serve as a general platform for microfluidic processing.

Referring now to FIGS. 4A-7B, examples of some basicdroplet-manipulative operations are illustrated. As in the case of FIG.1, a linear arrangement of three unit cells C₁, C₂ and C₃ and associatedcontrol electrodes E₁, E₂ and E₃ are illustrated, again with theunderstanding that these unit cells C₁, C₂ and C₃ and control electrodesE₁, E₂ and E₃ can form a section of a larger linear series, non-linearseries, or two-dimensional array of unit cells/control electrodes. Forconvenience, in FIGS. 4B-7B, corresponding control electrodes and unitcells are collectively referred to as control electrodes E₁, E₂ and E₃.Moreover, unit cells C₁, C₂, and C₃ can be physical entities, such asareas on a chip surface, or conceptual elements. In each of FIGS. 4A-7B,an active (i.e., energized) control electrode E₁, E₂, or E₃ is indicatedby designating its associated electrical lead line L₁, L₂, or L₃ “ON”,while an inactive (i.e., de-energized, floated, or grounded) controlelectrode E₁, E₂, or E₃ is indicated by designating its associatedelectrical lead line L₁, L₂, or L₃ “OFF”.

Turning to FIGS. 4A-4D, a basic MOVE operation is illustrated. FIG. 4Aillustrates a starting position at which droplet D is centered oncontrol electrode E₁. Initially, all control electrodes E₁, E₂ and E₃are grounded so that droplet D is stationary and in equilibrium oncontrol electrode E₁. Alternatively, control electrode E₁ could beenergized while all adjacent control electrodes (e.g., E₂) are groundedso as to initially maintain droplet D in a “HOLD” or “STORE” state, andthereby isolate droplet D from adjoining regions of an array where othermanipulative operations might be occurring on other droplets. To movedroplet D in the direction indicated by the arrow in FIGS. 4A-4B,control electrode E₂ is energized to attract droplet D and thereby causedroplet D to move and become centered on control electrode E₂, as shownin FIG. 4B. Subsequent activation of control electrode E₃, followed byremoval of the voltage potential at control electrode E₂, causes dropletD to move onto control electrode E₃ as shown in FIG. 4C. This sequencingof electrodes can be repeated to cause droplet D to continue to move inthe desired direction indicated by the arrow. It will also be evidentthat the precise path through which droplet D moves across the electrodearray is easily controlled by appropriately programming an electroniccontrol unit (such as a conventional microprocessor) to activate andde-activate selected electrodes of the array according to apredetermined sequence. Thus, for example, droplet D can be actuated tomake right- and left-hand turns within the array. For instance, afterdroplet D has been moved to control electrode E₂ from E₁ as shown inFIG. 4B, droplet D can then be moved onto control electrode E₅ ofanother row of electrodes E₄-E₆ as shown in FIG. 4D. Moreover, droplet Dcan be cycled back and forth (e.g., shaken) along a desired number ofunit cells and at a desired frequency for various purposes such asagitation of droplet D, as described in the EXAMPLE hereinabove.

FIGS. 5A-5C illustrate a basic MERGE or MIX operation wherein twodroplets D₁ and D₂ are combined into a single droplet D₃. In FIG. 5A,two droplets D₁ and D₂ are initially positioned at control electrodes E₁and E₃ and separated by at least one intervening control electrode E₂.As shown in FIG. 5B, all three control electrodes E₁, E₂ and E₃ are thenactivated, thereby drawing droplets D₁ and D₂ toward each other acrosscentral control electrode E₂ as indicated by the arrows in FIG. 5B. Oncethe opposing sides of droplets D₁ and D₂ encounter each other at centralcontrol electrode E₂, a single meniscus M is created that joins the twodroplets D₁ and D₂ together. As shown in FIG. 5C, the two outer controlelectrodes E₁ and E₃ are then returned to the ground state, therebyincreasing the hydrophobicity of the surfaces of the unit cellsassociated with outer electrodes E₁ and E₃ and repelling the mergingdroplets D₁ and D₂, whereas energized central control electrode E₂increases the wettability of its proximal surface contacting droplets D₁and D₂. As a result, droplets D₁ and D₂ combine into a single mixeddroplet D₃ as shown in FIG. 5C, which represents the lowest energy statepossible for droplet D₃ under these conditions. The resulting combineddroplet D₃ can be assumed to have twice the volume or mass as either ofthe original, non-mixed droplets D₁ and D₂, since parasitic losses arenegligible or zero. This is because evaporation of the droplet materialis avoided due to the preferable use of a filler fluid (e.g., air or animmiscible liquid such as silicone oil) to surround the droplets,because the surfaces contacting the droplet material (e.g., upper andlower hydrophobic layers 27 and 23 shown in FIG. 1) are low-frictionsurfaces, and/or because the electrowetting mechanism employed by theinvention is non-thermal.

In the present discussion, the terms MERGE and MIX have been usedinterchangeably to denote the combination of two or more droplets. Thisis because the merging of droplets does not in all cases directly orimmediately result in the complete mixing of the components of theinitially separate droplets. Whether merging results in mixing candepend on many factors. These factors can include the respectivecompositions or chemistries of the droplets to be mixed, physicalproperties of the droplets or their surroundings such as temperature andpressure, derived properties of the droplets such as viscosity andsurface tension, and the amount of time during which the droplets areheld in a combined state prior to being moved or split back apart. As ageneral matter, the mechanism by which droplets are mixed together canbe categorized as either passive or active mixing. In passive mixing,the merged droplet remain on the final electrode throughout the mixingprocess. Passive mixing can be sufficient under conditions where anacceptable degree of diffusion within the combined droplet occurs. Inactive mixing, on the other hand, the merged droplet is then movedaround in some manner, adding energy to the process to effect completeor more complete mixing. Active mixing strategies enabled by the presentinvention are described hereinbelow.

It will be further noted that in the case where a distinct mixingoperation is to occur after a merging operation, these two operationscan occur at different sections or areas on the electrode array of thechip. For instance, two droplets can be merged at one section, and oneor more of the basic MOVE operations can be implemented to convey themerged droplet to another section. An active mixing strategy can then beexecuted at this other section or while the merged droplet is in transitto the other section, as described hereinbelow.

FIGS. 6A-6C illustrate a basic SPLIT operation, the mechanics of whichare essentially the inverse of those of the MERGE or MIX operation justdescribed. Initially, as shown in FIG. 6A, all three control electrodesE₁, E₂ and E₃ are grounded, so that a single droplet D is provided oncentral control electrode E₂ in its equilibrium state. As shown in FIG.6B, outer control electrodes E₁ and E₃ are then energized to drawdroplet D laterally outwardly (in the direction of the arrows) ontoouter control electrodes E₁ and E₃. This has the effect of shrinkingmeniscus M of droplet D, which is characterized as “necking” with outerlobes being formed on both energized control electrodes E₁ and E₃.Eventually, the central portion of meniscus M breaks, thereby creatingtwo new droplets D₁ and D₂ split off from the original droplet D asshown in FIG. 6C. Split droplets D₁ and D₂ have the same orsubstantially the same volume, due in part to the symmetry of thephysical components and structure of electrowetting microactuatormechanism 10 (FIG. 1), as well as the equal voltage potentials appliedto outer control electrodes E₁ and E₃. It will be noted that in manyimplementations of the invention, such as analytical and assayingprocedures, a SPLIT operation is executed immediately after a MERGE orMIX operation so as to maintain uniformly-sized droplets on themicrofluidic chip or other array-containing device.

Referring now to FIGS. 7A and 7B, a DISCRETIZE operation can be derivedfrom the basic SPLIT operation. As shown in FIG. 7A, a surface or portI/O is provided either on an electrode grid or at an edge thereofadjacent to electrode-containing unit cells (e.g., control electrodeE₁), and serves as an input and/or output for liquid. A liquiddispensing device 50 is provided, and can be of any conventional design(e.g., a capillary tube, pipette, fluid pen, syringe, or the like)adapted to dispense and/or aspirate a quantity of liquid LQ. Dispensingdevice 50 can be adapted to dispense metered doses (e.g., aliquots) ofliquid LQ or to provide a continuous flow of liquid LQ, either at portI/O or directly at control electrode E₁. As an alternative to usingdispensing device 50, a continuous flow of liquid LQ could be conductedacross the surface of a microfluidic chip, with control electrodes E₁,E₂, and E₃ being arranged either in the direction of the continuous flowor in a non-collinear (e.g., perpendicular) direction with respect tothe continuous flow. In the specific, exemplary embodiment shown in FIG.7A, dispensing device 50 supplies liquid LQ to control electrode E₁.

To create a droplet on the electrode array, the control electrodedirectly beneath the main body of liquid LQ (control electrode E₁) andat least two control electrodes adjacent to the edge of the liquid body(e.g., control electrodes E₁ and E₃) are energized. This causes thedispensed body of liquid LQ to spread across control electrodes E₁ andE₂ as shown in FIG. 7A. In a manner analogous to the SPLIT operationdescribed hereinabove with reference to FIGS. 6A-6C, the intermediatecontrol electrode (control electrode E₂) is then de-energized to createa hydrophobic region between two effectively hydrophilic regions. Theliquid meniscus breaks above the hydrophobic region to form or “pinchoff” a new droplet D, which is centered on control electrode E₃ as shownin FIG. 7B. From this point, further energize/de-energize sequencing ofother electrodes of the array can be effected to move droplet D in anydesired row-wise and/or column-wise direction to other areas on theelectrode array. Moreover, for a continuous input flow of liquid LQ,this dispensing process can be repeated to create a train of droplets onthe grid or array, thereby discretizing the continuous flow. Asdescribed in more detail hereinbelow, the discretization process ishighly useful for implementing droplet-based processes on the array,especially when a plurality of concurrent operations on many dropletsare contemplated.

Droplet-Based Mixing Strategies

Examples of several strategies for mixing droplets in accordance withthe present invention will now be described. Referring to FIGS. 8A and8B, a configuration such as that of electrowetting microactuatormechanism 10, described hereinabove with reference to FIG. 1, can beemployed to carry out merging and mixing operations on two or moredroplets, e.g., droplets D₁ and D₂. In FIGS. 8A and 8B, droplets D₁ andD₂ are initially centrally positioned on control electrodes E₂ and E₅,respectively. Droplets D₁ and D₂ can be actuated by electrowetting tomove toward each other and merge together on a final electrode in themanner described previously with reference to FIGS. 5A-5C. The finalelectrode can be an intermediately disposed electrode such as electrodeE₃ or E₄. Alternatively, one droplet can move across one or more controlelectrodes and merge into another stationary droplet. Thus, asillustrated in FIGS. 8A and 8B, droplet D₁ can be actuated to moveacross intermediate electrodes E₃ and E₄ as indicated by the arrow andmerge with droplet D₂ residing on electrode, such that the merging ofdroplets D₁ and D₂ occurs on electrode E₅. The combined droplet can thenbe actively mixed according to either a one-dimensional linear,two-dimensional linear, or two-dimensional loop mixing strategy.

As one example of a one-dimensional linear mixing strategy, multipledroplets can be merged as just described, and the resulting combineddroplet then oscillated (or “shaken” or “switched”) back and forth at adesired frequency over a few electrodes to cause perturbations in thecontents of the combined droplet. This mixing process is described inthe EXAMPLE set forth hereinabove and can involve any number of linearlyarranged electrodes, such as electrodes in a row or column of an array.FIGS. 9A, 9B and 9C illustrate two-, three-, and four-electrode series,respectively, in which merging and mixing by shaking can be performed.As another example of one-dimensional linear mixing, multiple dropletsare merged, and the combined droplet or droplets are then split apart asdescribed hereinabove. The resulting split/merged droplets are thenoscillated back and forth at a desired frequency over a few electrodes.The split/merged droplets can then be recombined, re-split, andre-oscillated for a number of successive cycles until the desired degreeof mixing has been attained. Both of these one-dimensional, linearmixing approaches produce reversible flow within the combined droplet ordroplets. It is thus possible that the mixing currents established bymotion in one direction could be undone or reversed when the combineddroplet oscillates back the other way. Therefore, in some situations,the reversible flow attending one-dimensional mixing processes mayrequire undesirably large mixing times.

Referring now to FIGS. 10A-10C, another example of one-dimensionallinear mixing referred to as “mixing-in-transport” is illustrated. Thismethod entails combining two or more droplets and then continuouslyactuating the combined droplet in a forward direction along a desiredflow path until mixing is complete. Referring to FIG. 10A, a combineddroplet D is transported from a starting electrode E_(o) along aprogrammed path of electrodes on the array until it reaches apreselected destination electrode E_(f). Destination electrode E_(f) canbe a location on the array at which a subsequent process such asanalysis, reaction, incubation, or detection is programmed to occur. Insuch a case, the flow path over which combined droplet D is activelymixed, indicated by the arrow, also serves as the analysis flow pathover which the sample is transported from the input to the processingarea on the array. The number of electrodes comprising the selected pathfrom starting electrode E_(o) to destination electrode E_(f) correspondsto the number of actuations to which combined droplet D is subject.Hence, through the use of a sufficient number of intermediate pathelectrodes, combined droplet D will be fully mixed by the time itreaches destination electrode E_(f). It will be noted that the flow pathdoes not reverse as in the case of the afore-described oscillatorymixing techniques. The flow path can, however include one or moreright-angle turns through the x-y plane of the array as indicated by therespective arrows in FIGS. 10A-10C. In some cases, turning the pathproduces unique flow patterns that enhance the mixing effect. In FIG.10B, the flow path has a ladder or step structure consisting of a numberof right-angle turns. In FIG. 10C, destination electrode E_(f) lies inthe same row as starting electrode E_(o), but combined droplet D isactuated through a flow path that deviates from and subsequently returnsto that row in order to increase the number of electrodes over whichcombined droplet D travels and the number of turns executed.

Referring now to FIG. 11, an example of a two-dimensional linear mixingstrategy is illustrated. One electrode row E_(ROW) and one electrodecolumn E_(COL) of the array are utilized. Droplets D₁ and D₂ are movedtoward each other along electrode row E_(ROW) and merged as describedhereinabove, forming a merged droplet D₃ centered on the electrodedisposed at the intersection of electrode row E_(ROW) and electrodecolumn E_(COL). Selected electrodes of electrode column E_(COL) are thensequentially energized and de-energized in the manner describedhereinabove to split merged droplet D₃ into split droplets D₄ and D₅.Split droplets D₄ and D₅ are then moved along electrode column E_(COL).This continued movement of split droplets D₄ and D₅ enhances the mixingeffect on the contents of split droplets D₄ and D₅.

Referring now to FIGS. 12A and 12B, examples of two-dimensional loopmixing strategies are illustrated. In FIG. 12A, a combined droplet D iscirculated clockwise or counterclockwise in a circular, square or otherclosed loop path along the electrodes of selected rows and columns ofthe array, as indicated by the arrow. This cyclical actuation ofcombined droplet D is effected through appropriate sequencing of theelectrodes comprising the selected path. Combined droplet D is cycled inthis manner for a number of times sufficient to mix its contents. Thecycling of combined droplet D produces nonreversible flow patterns thatenhance the mixing effect and reduce the time required for completemixing. In FIG. 12A, the path circumscribes only one central electrodenot used for actuation, although the path could be made larger so as tocircumscribe more central electrodes.

In FIG. 12B, a sub-array of at least four adjacent electrodes E₁-E₄ isutilized. Combined droplet D is large enough to overlap all fourelectrodes E₁-E₄ of the sub-array simultaneously. The larger size ofcombined droplet D could be the result of merging two smaller-sizeddroplets without splitting, or could be the result of first merging twopairs of droplets and thereafter combining the two merged droplets.Combined droplet D is rotated around the sub-array by sequencingelectrodes E₁-E₄ in the order appropriate for effecting either clockwiseor counterclockwise rotation. As compared with the mixing strategyillustrated in FIG. 12A, however, a portion of the larger-sized combineddroplet D remains “pinned” at or near the intersection of the fourelectrodes E₁-E₄ of the sub-array. Thus, combined droplet D in effectrotates or spins about the intersecting region where the pinned portionis located. This effect gives rise to unique internal flow patterns thatenhance the mixing effect attributed to rotating or spinning combineddroplet D and that promote nonreversible flow. Moreover, the ability tomix combined droplet D using only four electrodes E₁-E₄ enables thecyclical actuation to occur at high frequencies and with less powerrequirements.

The mixing strategy illustrated in FIG. 12B can also be implementedusing other sizes of arrays. For instance, a 2×4 array has been found towork well in accordance with the invention.

For all of the above-described mixing strategies, it will be noted thedroplets involved can be of equal size or unequal volumes. In asituation where an n:1 volume ratio of mixing is required, the electrodeareas can be proportionately chosen to yield a one-droplet (n) toone-droplet (1) mixing.

FIG. 13 depicts graphical data illustrating the performance of theone-dimensional linear mixing strategy. The time for complete mixing isplotted as a function of frequency of droplet oscillation (i.e., theswitch time between one electrode and a neighboring electrode). Curvesare respectively plotted for the 2-electrode (see FIG. 9A), 3-electrode(see FIG. 9B), and 4-electrode (see FIG. 9C) mixing configurations.Mixing times were obtained for 1, 2, 4, 8, and 16 Hz frequencies. Theactuation voltage applied to each electrode was 50 V. It was observedthat increasing the frequency of switching results in faster mixingtimes. Similarly, for a given frequency, increasing the number ofelectrodes also results in improved mixing. It was concluded thatincreasing the number of electrodes on which the oscillation of themerged droplets is performed increases the number of multi-laminateconfigurations generated within the droplet, thereby increasing theinterfacial area available for diffusion.

FIG. 14 depicts graphical data illustrating the performance of thetwo-dimensional loop mixing strategy in which the droplet is largeenough to overlap the 2×2 electrode sub-array (see FIG. 12B). Mixingtimes were obtained for 8, 16, 32, and 64 Hz frequencies. As in theexperiment that produced the plot of FIG. 13, the actuation voltageapplied to each electrode was 50 V. It was concluded thattwo-dimensional mixing reduces the effect of flow reversibilityassociated with one-dimensional mixing. Moreover, the fact that thedroplet rotates about a point enabled the switching frequency to beincreased up to 64 Hz for an actuation voltage of 50 V. This frequencywould not have been possible in a one-dimensional linear actuation caseat the same voltage. It is further believed that the fact that thedroplet overlaps all four electrodes simultaneously enabled droplettransport at such high frequencies and low voltages. The time betweenthe sequential firing of any two adjacent electrodes of the 2×2sub-array can be reduced because the droplet is in electricalcommunication with both electrodes simultaneously. That is, the lag timeand distance needed for the droplet to physically move from oneelectrode to another is reduced. Consequently, the velocity of thedroplet can be increased in the case of two-dimensional mixing, allowingvortices to form and thereby promoting mixing.

Droplet-Based Sampling and Processing

Referring now to FIGS. 15A and 15B, a method for sampling andsubsequently processing droplets from a continuous-flow fluid inputsource 61 is schematically illustrated in accordance with the invention.More particularly, the method enables the discretization ofuniformly-sized sample droplets S from continuous-flow source 61 bymeans of electrowetting-based techniques as described hereinabove, inpreparation for subsequent droplet-based, on-chip and/or off-chipprocedures (e.g., mixing, reacting, incubation, analysis, detection,monitoring, and the like). In this context, the term “continuous” istaken to denote a volume of liquid that has not been discretized intosmaller-volume droplets. Non-limiting examples of continuous-flow inputsinclude capillary-scale streams, fingers, slugs, aliquots, and metereddoses of fluids introduced to a substrate surface or other plane from anappropriate source or dispensing device. Sample droplets S willtypically contain an analyte substance of interest (e.g., apharmaceutical molecule to be identified such as by mass spectrometry,or a known molecule whose concentration is to be determined such as byspectroscopy). The several sample droplets S shown in FIGS. 15A and 15Brepresent either separate sample droplets S that have been discretizedfrom continuous-flow source 61, or a single sample droplet S movable todifferent locations on the electrode array over time and along variousanalysis flow paths available in accordance with the sequencing of theelectrodes.

The method can be characterized as digitizing analytical signals from ananalog input to facilitate the processing of such signals. It will beunderstood that the droplet-manipulative operations depicted in FIGS.15A and 15B can advantageously occur on an electrode array as describedhereinabove. Such array can be fabricated on or embedded in the surfaceof a microfluidic chip, with or without other features or devicesordinarily associated with IC, MEMS, and microfluidic technologies.Through appropriate sequencing and control of the electrodes of thearray such as through communication with an appropriate electroniccontroller, sampling (including droplet formation and transport) can bedone on a continuous and automated basis.

In FIG. 15A, the liquid input flow of continuous-flow source 61 issupplied to the electrode array at a suitable injection point. Utilizingthe electrowetting-based techniques described hereinabove, continuousliquid flow 61 is fragmented or discretized into a series or train ofsample droplets S of uniform size. One or more of these newly formedsample droplets S can then be manipulated according to a desiredprotocol, which can include one or more of the fundamental MOVE, MERGE,MIX and/or SPLIT operations described hereinabove, as well as anyoperations derived from these fundamental operations. In particular, theinvention enables sample droplets S to be diverted from continuousliquid input flow 61 for on-chip analysis or other on-chip processing.For example, FIG. 15A shows droplets being transported alongprogrammable analysis flow paths across the microfluidic chip to one ormore functional cells or regions situated on the surface of microfluidicchip such as cells 63 and 65.

Functional cells 63 and 65 can comprise, for example, mixers, reactors,detectors, or storage areas. In the case of mixers and reactors, sampledroplets S are combined with additive droplets R₁ and/or R₂ that aresupplied from one or more separate reservoirs or injection sites on oradjacent to the microfluidic chip and conveyed across the microfluidicchip according to the electrowetting technique. In the case of mixers,additive droplets R₁ and/or R₂ can be other sample substances whosecompositions are different from sample droplets S. Alternatively, whendilution of sample droplets S is desired, additive droplets R₁ and/or R₂can be solvents of differing types. In the case of reactors, additivedroplets R₁ and/or R₂ can contain chemical reagents of differing types.For example, the electrode array or a portion thereof could be employedas a miniaturized version of multi-sample liquid handling/assayingapparatus, which conventionally requires the use of such largecomponents as 96-well microtitre plates, solvent bottles, liquidtransfer tubing, syringe or peristaltic pumps, multi-part valves, androbotic systems.

Functional cells 63 and 65 preferably comprise one or moreelectrode-containing unit cells on the array. Such functional cells 63and 65 can in many cases be defined by the sequencing of theircorresponding control electrodes, where the sequencing is programmed aspart of the desired protocol and controlled by an electronic controlunit communicating with the microfluidic chip. Accordingly, functionalcells 63 and 65 can be created anywhere on the electrode array of themicrofluidic chip and reconfigured on a real-time basis. For example,FIG. 16 illustrates a mixer cell, generally designated MC, that can becreated for mixing or diluting a sample droplet S with an additivedroplet R according to any of the mixing strategies disclosed herein.Mixer cell MC comprises a 5×3 matrix of electrode-containing unit cellsthat could be part of a larger electrode array provided by the chip.Mixer cell MC is thus rendered from five electrode/cell rows ROW1-ROW5and three electrode/cell columns COL1-COL3. MERGE and SPLIT operationscan occur at the centrally located electrodes E₁-E₃ as describedhereinabove with reference to FIGS. 5A-6C. The electrodes associatedwith outer columns COL1 and COL3 and outer rows ROW1 and ROW5 can beused to define transport paths over which sample droplet S and additivedroplet R are conveyed from other areas of the electrode array, such asafter being discretized from continuous-flow source 61 (see FIG. 15A or15B). A 2×2 sub-array can be defined for implementing two-dimensionalloop mixing processes as illustrated in FIG. 12B. During a MIX, MERGE,SPLIT, or HOLD operation, some or all of the electrodes associated withouter columns COL1 and COL3 and outer rows ROW1 and ROW5 can be groundedto serve as gates and thus isolate mixer cell MC from other areas on thechip. If necessary, complete or substantially complete mixing can beaccomplished by a passive mechanism such as diffusion, or by an activemechanism such as by moving or “shaking” the combined droplet accordingto electrowetting as described hereinabove.

The invention contemplates providing other types of functional cells,including functional cells that are essentially miniaturized embodimentsor emulations of traditional, macro-scale devices or instruments such asreactors, detectors, and other analytical or measuring instruments. Forexample, a droplet could be isolated and held in a single row or columnof the main electrode array, or at a cell situated off the main array,to emulate a sample holding cell or flow cell through which a beam oflight is passed in connection with known optical spectroscopictechniques. A light beam of an initial intensity could be provided froman input optical fiber and passed through the droplet contained by thesample cell. The attenuated light beam leaving the droplet could thenenter an output optical fiber and routed to an appropriate detectionapparatus such as a photocell. The optical fibers could be positioned oneither side of the sample cell, or could be provided in a miniature dipprobe that is incorporated with or inserted into the sample cell.

Referring back to FIG. 15A, upon completion of a process executed at afunctional cell (e.g., cell 63 or 65), the resulting product droplets(not shown) can be conveyed to respective reservoirs 67 or 69 locatedeither on or off the microfluidic chip for the purpose of wastecollection, storage, or output. In addition, sample droplets S and/orproduct droplets can be recombined into a continuous liquid output flow71 at a suitable output site on or adjacent to the microfluidic chip forthe purposes of collection, waste reception, or output to a furtherprocess. Moreover, the droplets processed by functional cell 63 or 65can be prepared sample droplets that have been diluted and/or reacted inone or more steps, and then transported by electrowetting to anotherportion of the chip dedicated to detection or measurement of theanalyte. Some detection sites can, for example, contain bound enzymes orother biomolecular recognition agents, and be specific for particularanalytes. Other detection sites can consist of a general means ofdetection such as an optical system for fluorescence- orabsorbance-based assays, an example of which is given hereinabove.

In the alternative embodiment shown in FIG. 15B, continuous liquid flow61 is supplied from an input site 61A, and completely traverses thesurface of the microfluidic chip to an output site 61B. In thisembodiment, sample droplets S are formed (i.e., continuous liquid inputflow 61 is sampled) at specific, selectable unit-cell locations alongthe length of continuous liquid input flow 61 such as the illustratedlocation 73, and subsequent electrowetting-based manipulations areexecuted as described hereinabove in relation to the embodiment of FIG.15A.

The methods described in connection with FIGS. 15A and 15B have utilityin many applications. Applications of on-line microfluidic analysis caninclude, for example, analysis of microdialysis or other biologicalperfusion flows, environmental and water quality monitoring andmonitoring of industrial and chemical processes such as fermentation.Analysis can include the determination of the presence, concentration oractivity of any specific substance within the flowing liquid. On-linecontinuous analysis is beneficial in any application where real-timemeasurement of a time-varying chemical signal is required, a classicexample being glucose monitoring of diabetic patients. Microfluidicsreduces the quantity of sample required for an analysis, therebyallowing less invasive sampling techniques that avoid depleting theanalyte being measured, while also permitting miniaturized and portableinstruments to be realized.

The droplet-based methods of the invention provide a number ofadvantages over known continuous flow-based microscale methods as wellas more conventional macroscale instrument-based methods. Referring toeither FIG. 15A or 15B, the flow of sample droplets S fromcontinuous-flow source 61 to the analysis portion of the chip (i.e., theanalysis flow) is controlled independently of the continuous flow (i.e.,the input flow), thereby allowing a great deal of flexibility incarrying out the analyses. The de-coupling of the analysis flow from thecontinuous input flow allows each respective flow to be separatelyoptimized and controlled. For example, in microdialysis, the continuousflow can be optimized to achieve a particular recovery rate while theanalysis flow is optimized for a particular sensitivity or samplingrate. Reagent droplets R can be mixed with sample droplets S in theanalysis flow without affecting or contaminating the main input flow.Sample droplets S in the analysis flow can be stored or incubatedindefinitely without interrupting the input flow. Analyses requiringdifferent lengths of time can be carried out simultaneously and inparallel without interrupting the input flow.

In either embodiment depicted in FIG. 15A or 15B, the analysis or otherprocessing of sample droplets S is carried out on-line insofar as theanalysis occurs as part of the same sequential process as the input ofcontinuous-flow source 61. However, the analysis is not carried outin-line with respect to continuous liquid input flow 61, because newlyformed sample droplets S are diverted away from continuous liquid inputflow 61. This design thus allows the analysis flow to be de-coupled fromthe input flow.

As another advantage, multiple analytes can be simultaneously measured.Since continuous liquid flow 61 is fragmented into sample droplets S,each sample droplet S can be mixed with a different reagent droplet R₁or R₂ or conducted to a different test site on the chip to allowsimultaneous measurement of multiple analytes in a single sample withoutcross-talk or cross-contamination. Additionally, multiple step chemicalprotocols are possible, thereby allowing a wide range of types ofanalyses to be performed in a single chip.

Moreover, calibration and sample measurements can be multiplexed.Calibrant droplets can be generated and measured between samples.Calibration does not require cessation of the input flow, and periodicrecalibration during monitoring is possible. In addition, detection orsensing can be multiplexed for multiple analytes. For example, a singlefluorimeter or absorbance detector may be utilized to measure multipleanalytes by sequencing the delivery of sample droplets S to the detectorsite.

Another important advantage is the reconfigurability of the operation ofthe chip. Sampling rates can be dynamically varied through softwarecontrol. Mixing ratios, calibration procedures, and specific tests canall be controlled through software, allowing flexible and reconfigurableoperation of the chip. Feedback control is possible, which allowsanalysis results to influence the operation of the chip.

Droplet-Based Binary Interpolating Digital Mixing

Referring now to FIG. 17, a binary mixing apparatus, generallydesignated 100, is illustrated in accordance with the invention. Binarymixing apparatus 100 is useful for implementing a droplet-based,variable dilution binary mixing technique in one, two or more mixingphases to obtain desired mixing ratios. The degree of precision of theresulting mixing ratio depends on the number of discrete binary mixingunits utilized. As one example, FIG. 17 schematically illustrates afirst binary mixing unit 110 and a second binary mixing unit 210. Whenmore than one mixing unit is provided, a buffer 310 is preferablyprovided in fluid communication with the mixing units to storeintermediate products and transfer intermediate products between themixing units as needed. A suitable electronic controller EC such as amicroprocessor capable of executing the instructions of a computerprogram communicates with first binary mixing unit 110, second binarymixing unit 210, and buffer 310 through suitable communication lines111, 211, and 311, respectively.

Binary mixing apparatus 100 can be fabricated on a microfluidic chip forthe purpose of carrying out binary interpolating digital mixingprocedures in accordance with the invention. In designing the physicallayouts of the various droplet-handling components of binary mixingapparatus 100 (examples of which are illustrated in FIGS. 18A and 20),electrode design and transportation design (scheduling) were considered.The particular physical layout at least in part determines the code orinstruction set executed by electronic controller EC to control theelectrodes and thus the types and sequences of droplet-basedmanipulation to be performed. Preferably, the electrode-containingdroplet-handling regions of binary mixing apparatus 100 are structuredas shown in the cross-sectional view of FIG. 1, described hereinabove inconnection with electrowetting microactuator mechanism 10, or accordingto a single-sided electrode configurations described hereinbelow. Theelectrodes of each mixing unit can be sequenced to implement any of themixing strategies disclosed herein.

The architecture of binary mixing apparatus 100 is designed to take fulladvantage of accelerated rates observed in droplet-to-droplet mixingexperiments, while allowing precisely controlled mixing ratios that canbe varied dynamically for multi-point calibrations. As will becomeevident from the description herein, binary mixing apparatus 100 canhandle a wide range of mixing ratios with certain accuracy, and enablesmixing patterns that demonstrate high parallelism in the mixingoperation as well as scalability in the construction of mixingcomponents in a two-dimensional array. Binary mixing apparatus 100 canhandle a wide range of droplet sizes. There is, however, a lower limiton droplet size if sample droplets are being prepared for the purpose ofa detection or measurement.

The architecture of binary mixing apparatus 100 is based on therecognition that the most efficient mixing most likely occurs betweentwo droplets moving toward each other. This has been observed fromexperiments, and could be explained by the fact that convection inducedby shear movement of fluids accelerates the mixing process much fasterthan pure physical diffusion. Thus, as a general design principle,one-by-one mixing is utilized as much as possible. As indicatedhereinabove, one-by-one mixing preferably involves both mixing andsplitting operations to maintain uniform droplet size. The basic MIX andSPLIT operations have been described hereinabove with reference to FIGS.5A-6C.

Certain assumptions have been made in design of the architecture ofbinary mixing apparatus 100, and include the following:

1. Full mixing occurs in terms of chemical and/or physical processesgiven adequate time.

2. Equal droplet splitting occurs in terms of physical volume andchemical components.

3. Negligible residues are produced during droplet transportation.

4. Mixing time for large dilution ratios is a bottleneck.

5. There are tolerances on mixing ratios.

6. Transportation time is negligible compared to mixing.

Preferred design requirements and constraints were also considered, andinclude the following:

1. Minimum volume of mixture output to guarantee detectability.

2. Maximum number of independent control electrodes.

3. Maximum mixing area.

4. Maximum number of actuation per electrode.

5. Reconfigurability for different mixing ratios.

Thus, one design objective was to complete the mixing process using aminimum number of mixing-splitting operations while maintaining theaccuracy of the mixing ratio.

Moreover, some desirable attributes for an ideal mixing architecturewere considered to be as follows:

1. Accurate mixing ratio.

2. Small number of mixing cycles. Since many mixing processes willinvolve more than one mixing phase, during the first phase the twobinary mixing units 110 and 210 are operated in parallel to andindependent of each other. The second mixing phase, however, can onlystart after the first phase is finished. Thus, the total mixing time oftwo-phase mixing should be the maximum mixing time of first and secondbinary mixing units 110 and 210 in the first phase plus the mixing timeof either first binary mixing unit 110 or second binary mixing unit 210in the second phase. Accordingly, the mixing cycle is defined as thetotal mixing time required to finish one mixing process. It isstandardized in terms of mixing operations, which are assumed to be themost time consuming operations as compared to, for example, droplettransport.

3. Small number of total mixing operations. A single binary mixingoperation that consists of mixing, splitting and/or transportation is asource of error. Also, more mixing operations also mean more usage ofthe electrodes, which may be another cause of error due to the chargeaccumulation on electrodes.

4. Simplicity of operations.

5. Scalability. The capability of the binary mixing apparatus 100 tohandle different mixing ratios and extendibility of the structure tomultiple mixing units when large throughput is demanded.

6. Parallelism.

The architecture of binary mixing apparatus 100 implements multiplehierarchies of binary mixing phases, with the first hierarchy providingthe approximate mixing ratio and the following ones employed as thecalibration mechanism. The concept is analogous to an interpolatingDigital-to-Analog Converter (DAC) whose architecture is divided into twoparts, with the main DAC handling the MSB (most significant bit) in abinary manner and the sub-DAC dealing with calibration and correctiondown to the LSB (least significant bit). An example of a one-phasebinary mixing process carried out to produce sixteen sample dropletsdiluted to a concentration of 1/32 is described hereinbelow withreference to FIGS. 19A-19F.

It is believed that mixing in a binary manner results in dilution tolarge ratios in the power of two with only a few mixing operations. Theaccuracy of the ratio can be calibrated by further mixing twointermediate products in a binary manner. For example, one mixingprocess could produce concentrations of 1/8, and another could produceconcentrations of 1/16. When these two mixtures further mix with 1:1,1:3, 3:1, 1:7, and 7:1 ratios, respectively, the final product wouldhave concentrations of 1/10.67, 1/12.8, 1/9.14, 1/14.2, and 1/8.53,respectively. Based on this principle, any ratio can be obtained in afew mixing phases with acceptable tolerance. If further accuracy isneeded, an additional mixing phase using products from the previousphase can be used to calibrate the ratio. As indicated previously, theprocess of approaching the expected ratio to high accuracy could becharacterized as a successive approximation process that is similar toone used in Analog to Digital converter design. It is an approach thattrades off speed with accuracy. However, the number of mixing phasesrequired for adequate accuracy is surprisingly small. Generally, whenthe required ratio is smaller than 32, two mixing phases are oftenenough. Ratios larger than 32 but smaller than 64 would possibly needthree mixing phases. It is also observed that different combinations ofintermediate products mixed with a range of binary ratios would producemore interpolating points to further increase the accuracy, thuseliminating the necessities of using extra mixing phases.

Based on known mathematical principles, the architecture of binarymixing apparatus 100 can be designed to have preferably twosame-structured mixing units (e.g., first binary mixing unit 110 andsecond binary mixing unit 210 shown in FIG. 17), with each binary mixingunit 110 and 210 handling binary mixing and generating certain volumesof mixture. Each binary mixing unit 110 and 210 can produce differentmixing ratios of a power of two according to different operations. Inthe first mixing phase, the sample is mixed with the reagent with aratio of any of the series (1:1, 1:3, 1:7 . . . 1:2^(n−1)) using twobinary mixing units 110 and 210 in parallel. The products are twomixtures with the same volume. The ratio of the two mixtures isdetermined by the required ratio of the final product, and preferably iscontrolled by a computer program. In a second phase, the two mixturesmix with a certain binary ratio in one of the two units. Buffer 310 isused to store some of the intermediate products when second phase mixingis carried out in one of binary mixing units 110 or 210. Since thevolume of the intermediate product is limited (e.g., 16 droplets), thesecond mixing cannot be carried out with an arbitrarily large binaryratio. From the description herein of the structure and operation ofbinary mixing apparatus 100, it can be demonstrated that the possiblebinary ratio in the following mixing phase is constrained to be lessthan or equal to 31, given that 4 columns and 16 droplets are generatedfrom each unit. Even so, sufficient accuracy could be obtained after asecond phase. If further accuracy is demanded, additional mixing can becarried out to generate a mixture closer to the requirement, using theproduct from the second phase and another mixture with power of twoseries ratio (e.g., a calibration mixture).

From the description above, it can be observed that generating powers oftwo series mixtures can be a fundamental process in obtaining anexpected ratio. The exact ratio of this mixture could be decided aheadof time or varied dynamically. For example, during the first phase ofmixing, the two ratios could be calculated ahead of time according tothe required ratio. In the phase following the second phase, however,the calibration mixture could be decided dynamically, given the feedbackfrom the quality of previous mixing. Even if predecided, it is likelythat extra time would be needed to prepare the calibration mixturebefore a further phase mixing is carried out. In such a case, the use ofonly two binary mixing units 110 and 210 might be not enough, and anextra binary mixing unit could be added to prepare the calibrationmixture in parallel with the previous calibration mixing process.

The determination of a mixing strategy includes calculating the numberof mixing phases and the mixing ratio for each phase according to therequired ratio and its tolerance. This determination can be solved by anoptimization process with the number of mixing operations and time ofthe mixing as the objective function.

Referring now to FIGS. 18A and 18B, an exemplary architecture for firstbinary mixing unit 110 is illustrated, with the understanding thatsecond binary mixing unit 210 and any other additional mixing unitsprovided can be similarly designed. The embodiment shown in FIG. 18A iscapable of one-phase mixing, while the embodiment shown in FIG. 20 (tobe briefly described hereinbelow) is capable of two-phase mixing. Asshown in FIG. 18A, first binary mixing unit 110 generally comprises a7×7 electrode matrix or array, generally designated EA, consisting of 49matrix electrodes and their associated cells E_(ij), where “i”designates 1, 2, . . . , 7 rows of electrodes and “j” designates 1, 2, .. . , 7 columns of electrodes. FIG. 18B identifies matrix electrodesE_(ij) of electrode array EA in accordance with a two-dimensional systemof rows ROW1-ROW7 and columns COL1-COL7. The invention, however, is notlimited to any specific number of electrodes, rows, and columns. Alarger or smaller electrode array EA could be provided as appropriate.

Referring back to FIG. 18A, a sample reservoir 113, waste reservoir 115,and reagent reservoir 117 are also provided. Depending on the positionof reservoirs 113, 115 and 117 in relation to electrode array EA, asuitable number and arrangement of transport or path electrodes andassociated cells T₁-T₄ are provided for conveying droplets to and fromelectrode array EA. A number of electrical leads (e.g., L) are connectedto matrix electrodes E_(ij) and transport electrodes T₁-T₄ to controlthe movement or other manipulation of droplets. It will be understoodthat electrical leads L communicate with a suitable electroniccontroller such as a microprocessor (e.g., electronic controller EC inFIG. 17). Each matrix electrode E_(ij) could have its own independentelectrical lead connection. However, to reduce the number of electricalleads L and hence simplify the architecture of first binary mixing unit110, the electrodes of each of columns COL2-COL7 (see FIG. 18B) areconnected to common electrical leads L as shown in FIG. 18A. Thesecommon connections must be taken into consideration when writing theprotocol for mixing operations to be carried out by first binary mixingunit 110.

In effect, each binary mixing unit 110 and 210 of binary mixingapparatus 100 is designed to have 4×4 logic cells with each cell storingthe sample, reagent or intermediate mixture. This can be conceptualizedby comparing the matrix layout of FIG. 18B with the 4×4 logic cellmatrix illustrated in FIGS. 19A-19F. The 4×4 construct accounts for thefact that droplets combine on intermediate control electrodes fromadjacent control electrodes (e.g., intermediate control electrode E₂ andadjacent control electrodes E₁ and E₃ in FIGS. 5A-6C), the mixed dropletis then split, and the newly formed mixed droplets are then returned tothe adjacent control electrodes at the completion of the MIX (orMIX-SPLIT) operation. Hence, certain rows of electrodes need only beused as temporary intermediate electrodes during the actual dropletcombination event. The construct also accounts for the fact that certaincolumns of electrodes need only be used for droplet transport (e.g.,shifting droplets from one column to another to make room for theaddition of new reagent droplets). In view of the foregoing, electroderows ROW2, ROW4 and ROW6, and columns COL2, COL4 and COL6 in FIG. 18Bare depicted simply as lines in FIGS. 19A-19F. Also in FIGS. 19A-19F,active electrodes are indicated by shaded bars, mixing operations areindicated by the symbol “- - - -> <- - - -”, and transport operationsare indicated by the symbol “- - - ->”. Additionally, dropletconcentrations are indicated by numbers (e.g., 0, 1, ½) next to rows andcolumns where droplets reside.

It can be seen that one-by-one mixing can occur between some of theadjacent cells in horizontal or vertical directions (from theperspective of the drawing sheets containing FIGS. 19A-19F), dependingon whether active electrodes exist between the two cells. In the firstcolumn, between any of the two adjacent row cells containing droplets,an active electrode exists that allows the two adjacent row cells toperform mixing operations. In other columns, there are no activeelectrodes between two row cells. This is illustrated, for example, inFIG. 19A. Between any of the columns containing droplets, electrodesexist that allow any of the cells in one column to conduct a mixingoperation with the cells of its adjacent column simultaneously. This isillustrated, for example, in FIG. 19D. By the use of the activeelectrodes, the content of a logic cell (i.e., a droplet) can move fromone row to another in the first column, or move between columns. Theemployment of the 4×4 logic structure is designed for the optimizationof binary operations, as demonstrated by the following example. It willbe noted that the volume output of the present one-mixing-unitembodiment of first binary mixing unit 110 is limited to 16 droplets,although the physical volume of the final product can be adjusted bychanging the size of each droplet.

To demonstrate how binary mixing apparatus 100 can produce any of thepower of two ratios, FIGS. 19A-19F illustrate an example of a series ofmixing operations targeting a 1:31 ratio (equal to 1/32 concentration).It can be seen that the mixing process has two basic stages: a row mixand a column mix. Generally, the purpose of the row mix is to approachthe range of the mixing ratio with a minimum volume of two mixinginputs. The purpose of the column mix is to produce the required volumeat the output and at the same time obtain another four-fold increase inratio. Thus, as indicated in FIGS. 19A-19F, to obtain a 1:31 ratio, therow mix results in a 1:7 ratio or 1/8 concentration (see FIG. 19D). Thecolumn mix assists in achieving the final product ratio of 1:31 or 1/32concentration (see FIG. 19F).

Referring specifically to FIG. 19A, a single row mix is performed bycombining a sample droplet S₁ having a concentration of 1 (i.e., 100%)with a reagent (or solvent) droplet R₁ having a concentration of 0. Thisresults in two intermediate-mixture droplets I₁ and I₂, each having a1/2 concentration as shown in FIG. 19B. One of the intermediate-mixturedroplets (e.g., I₁) is discarded, and a new reagent droplet R₂ is movedto the logic cell adjacent to the remaining intermediate-mixture droplet(e.g., I₂). Another row mix is performed by combiningintermediate-mixture droplet I₂ and reagent droplet R₂. This results twointermediate-mixture droplets I₃ and I₄, each having a 1/4 concentrationas shown in FIG. 19C. Two new reagent droplets R₃ and R₄ are then addedand, in a double row mix operation, combined with respectiveintermediate-mixture droplets I₃ and I₄. This results in fourintermediate-mixture droplets I₅-I₈, each having a 1/8 concentration asshown in FIG. 19D.

As also shown in FIG. 19D, four new reagent droplets R₅-R₈ are thenmoved onto the matrix adjacent to respective intermediate-mixturedroplets I₅-I₈. A column mix is then performed as between eachcorresponding pair of intermediate-mixture droplets I₅-I₈ and reagentdroplets R₅-R₈. This produces eight intermediate-mixture dropletsI₉-I₁₆, each having a 1/16 concentration as shown in FIG. 19E. As alsoshown in FIG. 19E, each column of four intermediate-mixture droplets,I₉-I₁₂ and I₁₃-I₁₆, respectively, is shifted over one column to theright to enable two columns of new reagent droplets, R₉-R₁₂ and R₁₃-R₁₆,respectively, to be loaded onto the outer columns of the matrix. Eachcorresponding pair of intermediate-mixture droplets and reagent droplets(e.g., I₉ and R₉, I₁₀ and R₁₀, etc.) is then combined through additionalcolumn mix operations.

As a result of these mixing operations, sixteen final-mixture productdroplets P₁-P₁₆ are produced, each having a final concentration of 1/32(corresponding to the target mix ratio of 1:31) as shown in FIG. 19F.Product droplets P₁-P₁₆ are now prepared for any subsequent operationcontemplated, such sampling, detection, analysis, and the like asdescribed by way of example hereinabove. Additionally, depending on theprecise mix ratio desired, product droplets P₁-P₁₆ can be subjected to asecond or even a third phase of mixing operations if needed as describedhereinabove. Such additional mixing phases can occur at a different areaon the electrode array of which first binary mixing unit 110 could be apart. Alternatively, as illustrated in FIG. 17, the final-mixturedroplets can be conveyed to another binary mixing apparatus (e.g.,second binary mixing unit 210) that fluidly communicates directly withfirst binary mixing unit 110 or through buffer 310.

The method of the invention can be applied to ratios less than orgreater than 31. For example, if the goal is to obtain a ratio of 1:15,the row mix would mix the input to a ratio of 1:3, which would requiretwo mixing operations instead of three for obtaining a mixing ratio of1:7. In terms of mixing operations, FIGS. 19A-19F can be used to showthat the first stage for row mix (single) and the discard operation forthe second stage could be eliminated in such a case.

To further explain the detailed operations for completing the mixing of1:31, a pseudo code for the example specifically illustrated in FIGS.19A-19F (and with general reference to FIG. 18B) is listed as follows:

-   -   1. Load S (1,1), Load R (2,1), Row Mix 1,2    -   2. (Discard (1,1), Load R (3,1)), Row Mix 2,3    -   3. Load R (1,1) Load R (3,1), (Row Mix 1,2, Row Mix 3,4)    -   4. Column Load R2, Column Mix 1,2    -   5. Column Move 2 to 3, Column Move 1 to 2, column Load R 1,        Column Load R4, (Column Mix 1,2 Column Mix 3,4)    -   6. Finish

The above pseudo code also standardizes the possible mixing operationsinto one mixing process. The sequence of the operations is subject tomore potential optimization to increase the throughput of the mixingwhile decreasing the number of mixing operations. This design also keepsin mind that the number of active electrodes should be maintained assmall as possible while making sure all the mixing operations functionproperly. In the preferred embodiment, each binary mixing unit 110 and210 (see FIG. 17) is designed to have 13 active electrodes to handle themixing functions. The capability of transporting the droplets into andinside the each binary mixing unit 110 and 210 is another consideration.Initially, the two outside columns of the array could be used astransportation channels running along both sides of the mixer to deliverdroplets into the mixer simultaneously with other operations of themixer. The same number of electrodes can also handle thesetransportation functions.

The second phase is the mixing process when the intermediate productsfrom two binary mixing units 110 and 210 (see FIG. 17) are to be mixed.It is similar to the standard binary mixing process in the first phasedescribed hereinabove with reference to FIGS. 19A-19F. The onlydifference is that the second-phase mixing is carried out in one ofbinary mixing units 110 and 210 holding the previous mixing product(e.g., product droplets P₁-P₁₆ shown in FIG. 19F). As indicatedpreviously, buffer 310 is used to hold some of the product during theprocess.

It can be calculated that the maximum ratio of mixing during the secondphase is limited to 31. The reason is that to obtain the maximum ratio,row mixing should be used as much as possible. When row mixing is usedto increase the ratio, less input is lost during the discard process.Thus, when there are finite amounts of input material, the first choiceis to see how far the row mixing can go until there is just enoughvolume left to fulfill the requirement for mixture output. In this way,it could be known that two mixtures with 16 droplets can only mix withthe largest ratio of 1:31 when the output requirement is specified to noless than 16 droplets. It can also be demonstrated from FIGS. 19A-19Fthat to mix with a ratio of 1:31, 16 droplets of reagents would be theminimum amount.

The physical layout for first binary mixing unit 110 illustrated in FIG.18A can be modified to better achieve two-phase mixing capability.Accordingly, referring now to FIG. 20, a two-phase mixing unit,generally designated 410, is illustrated. The architecture of two-phasemixing unit 410 is similar to that of first binary mixing unit 110 ofFIG. 18A, and thus includes the 7×7 matrix, a sample reservoir 413, awaste reservoir 415, a reagent reservoir 417, and an appropriate numberand arrangement of off-array electrodes as needed for transport ofdroplets from the various reservoirs to the 7×7 matrix. Two-phase mixingunit 410 additionally includes a cleaning reservoir 419 to supplycleaning fluid between mixing processes, as well as an outlet site 421for transporting product droplets to other mixing units or to buffer 310(see FIG. 17). Moreover, it can be seen that additional rows and columnsof electrodes are provided at the perimeter of the 7×7 matrix to providetransport paths for droplets to and from the matrix.

Further insight into the performance of the architecture of binarymixing apparatus 100 can be obtained by considering the TABLE set forthhereinbelow. This TABLE was constructed to list all the possibleinterpolating mixing ratios using a two-phase mixing strategy for amaximum mixing ratio of 63 (or, equivalently, a maximum concentration of1/64). The corresponding mixing parameters, such as the mixing ratio formixing unit 1 and 2 (e.g., first and second binary mixing units 110 and210) in the first phase, the mixing ratio for the second phase, and thetotal mixing cycles are also recorded. The TABLE can serve as a basisfor selecting the proper mixing strategy and/or further optimization interms of trading off accuracy with time, improving resource usage whenmultiple mixers exist, decreasing total mixing operations, improvingparallelism, and so on. The TABLE can be provided as a look-up table ordata structure as part of the software used to control apparatus 100.

The TABLE shows that there are a total of 196 mixing strategies usingthe architecture of the invention, which corresponds to 152 uniquemixing points. The 196 mixing strategies are calculated by interpolatingany possible combinations of two mixtures with power of two ratios under63. These points have non-linear instead of linear intervals. Thesmaller the ratio, the smaller the interval. The achievable points areplotted in FIG. 21. It is evident from the TABLE that the number ofachievable ratios is larger than traditional linear mixing points andthe distribution is more reasonable. In addition, certain volumes ofoutput other than one droplet can allow more tolerance on the errorcaused by one-by-one mixing. In terms of mixing cycles, the bestperformance is for mixing ratios of the power of two compared to theirnearby ratios. In terms of accuracy, the larger the ratio, generally theworse the performance, since a smaller number of interpolating pointscan be achieved.

It can be observed from the two-phase mixing plan plotted in FIG. 21that there are not enough points when the target ratio is larger than36. FIG. 21 shows that there is no point around a ratio of 40. Thedifference between the target and theoretical achievable ratio couldamount to 3. However, by careful examination of the achievable pointsaround 40, an appropriate usage of the remaining mixture from phase oneto further calibrate the available points can result in severaladditional interpolating points between 36.5714 and 42.6667, where thelargest error exists from the phase two mixing plan. For instance, themixing plan #183 in the TABLE calls for obtaining mixture 1 and mixture2 with ratios of 1:31 and 1:63, respectively, then mixing them with aratio of 3:1. It is known that there are ¾ parts of mixture 2 left. Soit is possible to mix the mixture from phase two with a concentration of36.517 with mixture 2 of concentration 63 using ratio of 3:1, 7:1, etc.That leads to a point at 40.9, 38.5, etc. In such manner, more accuracyis possible with an additional mixing phase, but with only a smallincrease in mixing cycles (two and three cycles, respectively, in thisexample), and at the expense of no additional preparation of calibrationmixture.

FIG. 22 demonstrates all the achievable points by one-phase, two-phase,and three-phase mixing plans. The total number of points is 2044. Thepoints achieved by phase three are obtained by using the product fromphase two and remaining products from phase one. They are calculated byconsidering the volume of the remaining product from phase one afterphase two has finished and reusing them to mix with products from phasetwo. The possible mixing ratios of phase three are determined by themixing ratio of phase two.

TABLE Mix Mix Mix Target Unit 1 Unit 2 Phase 2 Total Plan Mix Mix MixMix Mix Number Ratio Ratio Ratio Ratio Cycles 1 1.0159 1:0 1:1  31:1  62 1.0240 1:0 1:3  31:1  7 3 1.0281 1:0 1:7  31:1  8 4 1.0302 1:0 1:1531:1  9 5 1.0312 1:0 1:31 31:1  10 6 1.0317 1:0 1:63 31:1  11 7 1.03231:0 1:1  15:1  5 8 1.0492 1:0 1:3  15:1  6 9 1.0579 1:0 1:7  15:1  7 101.0622 1:0 1:15 15:1  8 11 1.0644 1:0 1:31 15:1  9 12 1.0656 1:0 1:6315:1  10 13 1.0667 1:0 1:1  7:1 4 14 1.1034 1:0 1:3  7:1 5 15 1.1228 1:01:7  7:1 6 16 1.1327 1:0 1:15 7:1 7 17 1.1378 1:0 1:31 7:1 8 18 1.14031:0 1:63 7:1 9 19 1.1429 1:0 1:1  3:1 3 20 1.2308 1:0 1:3  3:1 4 211.2800 1:0 1:7  3:1 5 22 1.3061 1:0 1:15 3:1 6 23 1.3196 1:0 1:31 3:1 724 1.3264 1:0 1:63 3:1 8 25 1.3333 1:0 1:1  1:1 2 26 1.6000 1:0 1:1  1:33 27 1.6000 1:0 1:3  1:1 3 28 1.7778 1:0 1:1  1:7 4 29 1.7778 1:0 1:7 1:1 4 30 1.8824 1:0 1:1   1:15 5 31 1.8824 1:0 1:15 1:1 5 32 1.9394 1:01:1   1:31 6 33 1.9394 1:0 1:31 1:1 6 34 1.9692 1:0 1:63 1:1 7 35 2.00001:1 N/A N/A 1 36 2.0317 1:1 1:3  31:1  7 37 2.0480 1:1 1:7  31:1  8 382.0562 1:1 1:15 31:1  9 39 2.0604 1:1 1:31 31:1  10 40 2.0624 1:1 1:6331:1  11 41 2.0645 1:1 1:3  15:1  6 42 2.0981 1:1 1:7  15:1  7 43 2.11571:1 1:15 15:1  8 44 2.1245 1:1 1:31 15:1  9 45 2.1289 1:1 1:63 15:1  1046 2.1333 1:1 1:3  7:1 5 47 2.2069 1:1 1:7  7:1 6 48 2.2456 1:1 1:15 7:17 49 2.2655 1:1 1:31 7:1 8 50 2.2756 1:1 1:63 7:1 9 51 2.2857 1:0 1:3 1:3 4 52 2.2857 1:1 1:3  3:1 4 53 2.4615 1:1 1:7  3:1 5 54 2.5600 1:11:15 3:1 6 55 2.6122 1:1 1:31 3:1 7 56 2.6392 1:1 1:63 3:1 8 57 2.66671:1 1:3  1:1 3 58 2.9091 1:0 1:3  1:7 5 59 2.9091 1:0 1:7  1:3 5 603.2000 1:1 1:3  1:3 4 61 3.2000 1:1 1:7  1:1 4 62 3.3684 1:0 1:3   1:156 63 3.3684 1:0 1:15 1:3 6 64 3.5556 1:1 1:3  1:7 5 65 3.5556 1:1 1:151:1 5 66 3.6571 1:0 1:3   1:31 7 67 3.6571 1:0 1:31 1:3 7 68 3.7647 1:11:3   1:15 6 69 3.7647 1:1 1:31 1:1 6 70 3.8209 1:0 1:63 1:3 8 71 3.87881:1 1:3   1:31 7 72 3.8788 1:1 1:63 1:1 7 73 4.0000 1:3 N/A N/A 3 744.0635 1:3 1:7  31:1  8 75 4.0960 1:3 1:15 31:1  9 76 4.1124 1:3 1:3131:1  10 77 4.1207 1:3 1:63 31:1  11 78 4.1290 1:3 1:7  15:1  7 794.1967 1:3 1:15 15:1  8 80 4.2314 1:3 1:31 15:1  9 81 4.2490 1:3 1:6315:1  10 82 4.2667 1:0 1:7  1:7 6 83 4.2667 1:3 1:7  7:1 6 84 4.4138 1:31:15 7:1 7 85 4.4912 1:3 1:31 7:1 8 86 4.5310 1:3 1:63 7:1 9 87 4.57141:1 1:7  1:3 5 88 4.5714 1:3 1:7  3:1 5 89 4.9231 1:3 1:15 3:1 6 905.1200 1:3 1:31 3:1 7 91 5.2245 1:3 1:63 3:1 8 92 5.3333 1:3 1:7  1:1 493 5.5652 1:0 1:7   1:15 7 94 5.5652 1:0 1:15 1:7 7 95 5.8182 1:1 1:7 1:7 6 96 5.8182 1:1 1:15 1:3 6 97 6.4000 1:3 1:7  1:3 5 98 6.4000 1:31:15 1:1 5 99 6.5641 1:0 1:7   1:31 8 100 6.5641 1:0 1:31 1:7 8 1016.7368 1:1 1:7   1:15 7 102 6.7368 1:1 1:31 1:3 7 103 7.1111 1:3 1:7 1:7 6 104 7.1111 1:3 1:31 1:1 6 105 7.2113 1:0 1:63 1:7 9 106 7.3143 1:11:7   1:31 8 107 7.3143 1:1 1:63 1:3 8 108 7.5294 1:3 1:7   1:15 7 1097.5294 1:3 1:63 1:1 7 110 7.7576 1:3 1:7   1:31 8 111 8.0000 1:7 N/A N/A4 112 8.1270 1:7 1:15 31:1  9 113 8.1920 1:7 1:31 31:1  10 114 8.22491:7 1:63 31:1  11 115 8.2581 1:0 1:15  1:15 8 116 8.2581 1:7 1:15 15:1 8 117 8.3934 1:7 1:31 15:1  9 118 8.4628 1:7 1:63 15:1  10 119 8.53331:1 1:15 1:7 7 120 8.5333 1:7 1:15 7:1 7 121 8.8276 1:7 1:31 7:1 8 1228.9825 1:7 1:63 7:1 9 123 9.1429 1:3 1:15 1:3 6 124 9.1429 1:7 1:15 3:16 125 9.8462 1:7 1:31 3:1 7 126 10.2400 1:7 1:63 3:1 8 127 10.6667 1:71:15 1:1 5 125 10.8936 1:0 1:15  1:31 9 129 10.8936 1:0 1:31  1:15 9 13011.1304 1:1 1:15  1:15 8 131 11.1304 1:1 1:31 1:7 8 132 11.6364 1:3 1:151:7 7 133 11.6364 1:3 1:31 1:3 7 134 12.8000 1:7 1:15 1:3 6 135 12.80001:7 1:31 1:1 6 136 12.9620 1:0 1:63  1:15 10 137 13.1282 1:1 1:15  1:319 138 13.1282 1:1 1:63 1:7 9 139 13.4737 1:3 1:15  1:15 8 140 13.47371:3 1:63 1:3 8 141 14.2222 1:7 1:15 1:7 7 142 14.2222 1:7 1:63 1:1 7 14314.6286 1:3 1:15  1:31 9 144 15.0588 1:7 1:15  1:15 8 145 15.5152 1:71:15  1:31 9 146 16.0000  1:15 N/A N/A 5 147 16.2540 1:0 1:31  1:31 10148 16.2540  1:15 1:31 31:1  10 149 16.3840  1:15 1:63 31:1  11 15016.5161 1:1 1:31  1:15 9 151 16.5161  1:15 1:31 15:1  9 152 16.7869 1:15 1:63 15:1  10 153 17.0667 1:3 1:31 1:7 8 154 17.0667  1:15 1:317:1 8 155 17:6552  1:15 1:63 7:1 9 156 18.2857 1:7 1:31 1:3 7 15718.2857  1:15 1:31 3:1 7 158 19.6923  1:15 1:63 3:1 8 159 21.3333  1:151:31 1:1 6 160 21.5579 1:0 1:63  1:31 11 161 21.7872 1:1 1:31  1:31 10162 21.7872 1:1 1:63  1:15 10 163 22.2609 1:3 1:31  1:15 9 164 22.26091:3 1:63 1:7 9 165 23.2727 1:7 1:31 1:7 8 166 23.2727 1:7 1:63 1:3 8 16725.6000  1:15 1:31 1:3 7 168 25.6000  1:15 1:63 1:1 7 169 26.2564 1:31:31  1:31 10 170 26.9474 1:7 1:31  1:15 9 171 28.4444  1:15 1:31 1:7 8172 29.2571 1:7 1:31  1:31 10 173 30.1176  1:15 1:31  1:15 9 174 31.0303 1:15 1:31  1:31 10 175 32.0000  1:31 N/A N/A 6 176 32.5079 1:1 1:63 1:31 11 177 32.5079  1:31 1:63 31:1  11 178 33.0323 1:3 1:63  1:15 10179 33.0323  1:31 1:63 15:1  10 180 34.1333 1:7 1:63 1:7 9 181 34.1333 1:31 1:63 7:1 9 182 36.5714  1:15 1:63 1:3 8 183 36.5714  1:31 1:63 3:18 184 42.6667  1:31 1:63 1:1 7 185 43.5745 1:3 1:63  1:31 11 186 44.52171:7 1:63  1:15 10 187 46.5455  1:15 1:63 1:7 9 188 51.2000  1:31 1:631:3 8 190 52.5128 1:7 1:63  1:31 11 191 53.8947  1:15 1:63  1:15 10 19256.8889  1:31 1:63 1:7 9 193 58.5143  1:15 1:63  1:31 11 194 60.2353 1:31 1:63  1:15 10 195 62.0606  1:31 1:63  1:31 11 196 64.0000  1:63N/A N/A 7

Electrowetting-Based Droplet Actuation on a Single-Sided Electrode Array

The aspects of the invention thus far have been described in connectionwith the use of a droplet actuating apparatus that has a two-sidedelectrode configuration such as microactuator mechanism 10 illustratedin FIG. 1. That is, lower plane 12 contains control or drive electrodesE₁-E₃ and upper plane 14 contains ground electrode G. As regardsmicroactuator mechanism 10, the function of upper plane 14 is to biasdroplet D at the ground potential or some other reference potential. Thegrounding (or biasing to reference) of upper plane 14 in connection withthe selective biasing of drive electrodes E₁-E₃ of lower plane 12generates a potential difference that enables droplet D to be moved bythe step-wise electrowetting technique described herein. However, inaccordance with another embodiment of the invention, the design of theapparatus employed for two-dimensional electrowetting-based dropletmanipulation can be simplified and made more flexible by eliminating theneed for a grounded upper plane 14.

Referring now to FIGS. 23A and 23B, a single-sided electrowettingmicroactuator mechanism, generally designated 500, is illustrated.Microactuator mechanism 500 comprises a lower plane 512 similar to thatof mechanism 10 of FIG. 1, and thus includes a suitable substrate 521 onwhich two-dimensional array of closely packed drive electrodes E (e.g.,drive electrodes E₁-E₃ and others) are embedded such as by patterning aconductive layer of copper, chrome, ITO, and the like. A dielectriclayer 523 covers drive electrodes E. Dielectric layer 523 ishydrophobic, and/or is treated with a hydrophobic layer (notspecifically shown). As a primary difference from microactuatormechanism 10 of FIG. 1, a two-dimensional grid of conducting lines G ata reference potential (e.g., conducting lines G₁-G₆ and others) has beensuperimposed on the electrode array of microactuator mechanism 500 ofFIGS. 23A and 23B, with each conducting line G running through the gapsbetween adjacent drive electrodes E. The reference potential can be aground potential, a nominal potential, or some other potential that islower than the actuation potential applied to drive electrodes E. Eachconducting line G can be a wire, bar, or any other conductive structurethat has a much narrower width/length aspect ratio in relation to driveelectrodes E. Each conducting line G could alternatively comprise aclosely packed series of smaller electrodes, but in most cases thisalternative would impractical due to the increased number of electricalconnections that would be required.

Importantly, the conducting line grid is coplanar or substantiallycoplanar with the electrode array. The conducting line grid can beembedded on lower plane 512 by means of microfabrication processescommonly used to create conductive interconnect structures onmicrochips. It thus can be seen that microactuator mechanism 500 can beconstructed as a single-substrate device. It is preferable, however, toinclude an upper plane 514 comprising a plate 525 having a hydrophobicsurface 527, such as a suitable plastic sheet or a hydrophobized glassplate. Unlike microactuator mechanism 10 of FIG. 1, however, upper plane514 of microactuator mechanism 500 of FIGS. 23A and 23B does notfunction as an electrode to bias droplet D. Instead, upper plane 514functions solely as a structural component to contain droplet D and anyfiller fluid such as an inert gas or immiscible liquid.

In the use of microactuator mechanism 500 for electrowetting-baseddroplet manipulations, it is still a requirement that a ground orreference connection to droplet D be maintained essentially constantlythroughout the droplet transport event. Hence, the size or volume ofdroplet D is selected to ensure that droplet D overlaps all adjacentdrive electrodes E as well as all conducting lines G surrounding thedrive electrode on which droplet D resides (e.g., electrode E₂ in FIG.23B). Moreover, it is preferable that dielectric layer 523 be patternedto cover only drive electrodes E so that conducting lines G are exposedto droplet D or at least are not electrically isolated from droplet D.At the same time, however, it is preferable that conducting lines G behydrophobic along with drive electrodes E so as not to impair movementof droplet D. Thus, in a preferred embodiment, after dielectric layer523 is patterned, both drive electrodes E and conducting lines G arecoated or otherwise treated so as render them hydrophobic. Thehydrophobization of conducting lines G is not specifically shown inFIGS. 23A and 23B. It will be understood, however, that the hydrophobiclayer covering conducting lines G is so thin that an electrical contactbetween droplet D and conducting lines G can still be maintained, due tothe porosity of the hydrophobic layer.

To operate microactuator mechanism 500, a suitable voltage source V andelectrical lead components are connected with conducting lines G anddrive electrodes E. Because conducting lines G are disposed in the sameplane as drive electrodes E, application of an electrical potentialbetween conducting lines G and a selected one of drive electrodes E₁,E₂, or E₃ (with the selection being represented by switches S₁-S₃ inFIG. 23A) establishes an electric field in the region of dielectriclayer 523 beneath droplet D. Analogous to the operation of microactuatormechanism 10 of FIG. 1, the electric field in turn creates a surfacetension gradient to cause droplet D overlapping the energized electrodeto move toward that electrode (e.g., drive electrode E₃ if movement isintended in right-hand direction in FIG. 23A). The electrode array canbe sequenced in a predetermined manner according to a set of softwareinstructions, or in real time in response to a suitable feedbackcircuit.

It will thus be noted that microactuator mechanism 500 with itssingle-sided electrode configuration can be used to implement allfunctions and methods described hereinabove in connection with thetwo-sided electrode configuration of FIG. 1, e.g., dispensing,transporting, merging, mixing, incubating, splitting, analyzing,monitoring, reacting, detecting, disposing, and so on to realize aminiaturized lab-on-a-chip system. For instance, to move droplet D shownin FIG. 23B to the right, drive electrodes E₂ and E₃ are activated tocause droplet D to spread onto drive electrode E₃. Subsequentde-activation of drive electrode E₂ causes droplet D to relax to a morefavorable lower energy state, and droplet D becomes centered on driveelectrode E₃. As another example, to split droplet D, drive electrodesE₁, E₂ and E₃ are activated to cause droplet D to spread onto driveelectrodes E₁ and E₃. Drive electrode E₂ is then de-activated to causedroplet D to break into two droplets respectively centered on driveelectrodes E₁ and E₃.

Referring now to FIGS. 24A-24D, an alternative single-sided electrodeconfiguration is illustrated in accordance with the present invention. Abase substrate containing an array of row and column biasing electrodesE_(ij) is again utilized as in previously described embodiments.Referring specifically to FIG. 24A, an array or portion of an array isshown in which three rows of electrodes E₁₁-E₁₄, E₂₁-E₂₅, and E₃₁-E₃₄,respectively, are provided. The rows and columns of the electrode arraycan be aligned as described herein for other embodiments of theinvention. Alternatively, as specifically shown in FIG. 24A, the arraycan be misaligned such that the electrodes in any given row are offsetfrom the electrodes of adjacent rows. For instance, electrodes E₁₁-E₁₄of the first row and electrodes E₃₁-E₃₄ of the third row are offset fromelectrodes E₂₁-E₂₅ of the intermediate second row. Whether aligned ormisaligned, the electrode array is preferably covered with insulatingand hydrophobic layers as in previously described embodiments. As in theconfiguration illustrated in FIGS. 23A and 23B, a top plate (not shown)can be provided for containment but does not function as an electrode.

In operation, selected biasing electrodes E_(ij) are dynamicallyassigned as either driving electrodes or grounding (or reference)electrodes. To effect droplet actuation, the assignment of a givenelectrode as a drive electrode requires that an adjacent electrode beassigned as a ground or reference electrode to create a circuitinclusive with droplet D and thereby enable the application of anactuation voltage. In FIG. 24A, electrode E₂₁ is energized and thusserves as the drive electrode, and electrode E₂₂ is grounded orotherwise set to a reference potential. All other electrodes E_(ij) ofthe illustrated array, or at least those electrodes surrounding thedriving/reference electrode pair E₂₁/E₂₂, remain in an electricallyfloating state. As shown in FIG. 24A, this activation causes droplet Doverlapping both electrodes E₂₁ and E₂₂ to seek an energeticallyfavorable state by moving so as to become centered along the gap orinterfacial region between electrodes E₂₁ and E₂₂.

In FIG. 24B, electrode E₂₁ is deactivated and electrode E₁₁ from anadjacent row is activated to serve as the next driving electrode.Electrode E₂₂ remains grounded or referenced. This causes droplet D tocenter itself between electrodes E₂₁, and E₂₂ by moving in a resultantnortheast direction, as indicated by the arrow. As shown in FIG. 24C,droplet D is actuated to the right along the gap between the first twoelectrode rows by deactivating electrode E₁₁ and activating electrodeE₁₂. As shown in FIG. 24D, electrode E₂₂ is disconnected from ground orreference and electrode E₂₃ is then grounded or referenced to causedroplet D to continue to advance to the right. It can be seen thatadditional sequencing of electrodes E_(ij) to render them either drivingor reference electrodes can be performed to cause droplet D to move inany direction along any desired flow path on the electrode array. It canbe further seen that, unlike previously described embodiments, the flowpath of droplet transport occurs along the gaps between electrodesE_(ij) as opposed to along the centers of electrodes E_(ij) themselves.It is also observed that the required actuation voltage will in mostcases be higher as compared with the configuration shown in FIGS. 23Aand 23B, because the dielectric layer covers both the driving andreference electrodes and thus its thickness is effectively doubled.

Referring now to FIGS. 25A and 25B, an electrode array with aligned rowsand columns can be used to cause droplet transport in straight lines ineither the north/south (FIG. 25A) or east/west (FIG. 25B) directions.The operation is analogous to that just described with reference toFIGS. 24A-24D. That is, programmable sequencing of pairs of drive andreference electrodes causes the movement of droplet D along the intendeddirection. In FIG. 25A, electrodes E₁₂, E₂₂ and E₃₂ of one column areselectively set to a ground or reference potential and electrodes E₁₃,E₂₃ and E₃₃ of an adjacent column are selectively energized. In FIG.25B, electrodes E₁₁, E₁₂, E₁₃ and E₁₄ of one row are selectivelyenergized and electrodes E₂₁, E₂₂, E₂₃ and E₂₄ of an adjacent row areselectively grounded or otherwise referenced.

It will be noted that a microactuator mechanism provided with thealternative single-sided electrode configurations illustrated in FIGS.24A-24D and FIGS. 25A and 25B can be used to implement all functions andmethods described hereinabove in connection with the two-sided electrodeconfiguration of FIG. 1. For instance, to split droplet D in either ofthe alternative configurations, three or more adjacent electrodes areactivated to spread droplet D and an appropriately selected interveningelectrode is then de-activated to break droplet D into two droplets.

It will be understood that various details of the invention may bechanged without departing from the scope of the invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation-the invention being defined by theclaims.

What is claimed is:
 1. An apparatus comprising: (a) a first substrateand a second substrate separated from one another to form a gaptherebetween, the gap comprising oil, wherein at least one of the firstsubstrate and second substrate comprises: (i) an array of driveelectrodes, wherein the array of drive electrodes are configured forconducting electrowetting mediated droplet operations in the gap; (ii) aconducting element comprising a conducting line grid comprisingelectrically intersecting conducting lines adjacent to the driveelectrodes, wherein the electrically intersecting conducting lines forma single electrical element set at a common reference potential; (iii) adielectric layer covering the drive electrodes, and a hydrophobic layercovering the drive electrodes and the conducting element, wherein thedielectric layer and the hydrophobic layer do not electrically isolatethe conducting element from a droplet when formed thereon; (b) anelectrode selector electrically configured to bias one or more selectedones of the drive electrodes of the array; (c) a first liquid supplyarea communicating with the array; (d) a second liquid supply areacommunicating with the array; and (e) drive electrodes locatedrespectively at said first sample liquid supply area and at said secondliquid supply area for being energized to form droplets from saidrespective liquid supply areas into the gap.
 2. The apparatus of claim 1wherein the electrode selector comprises a processor.
 3. The apparatusof claim 1 wherein the hydrophobic layer covering the conducting elementis sufficiently thin to permit electrical contact between the dropletand the conducting element.
 4. The apparatus of claim 1 wherein thedielectric layer has a permittivity and thickness, and the biasingcomprises applying a voltage to energize a drive electrode, and thepermittivity, thickness, and a voltage supply are selected to producedroplet movement by electrowetting for a droplet formed for conductingdroplet operations with the apparatus.
 5. The apparatus of claim 1wherein the conducting element is substantially coplanar with the arrayof drive electrodes.
 6. The apparatus of claim 1 wherein the conductingelement is coplanar with the array of drive electrodes.
 7. The apparatusof claim 1 wherein the conducting element comprises conductiveinterconnect structures.
 8. The apparatus of claim 1 wherein theconducting element comprises conducting lines positioned in relation toa first one of the drive electrodes such that a droplet atop the firstone of the drive electrodes having a size which causes it to partiallyoverlap an adjacent one of the drive electrodes electricallycommunicates an intervening conducting element.